Synthetic Immune Cells And Methods Of Use Thereof

Abstract

The present disclosure provides a genetically modified, in vitro immune cell. The immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator. The present disclosure provides compositions comprising the genetically modified, in vitro immune cell; and treatment methods comprising administration of the genetically modified, in vitro immune cell.

Description

CROSS-REFERENCING

[0001] This application claims the benefit of U.S. provisional application Ser. No. 62/901,999, filed on Sep. 18, 2019, which application is incorporated by reference herein.

INTRODUCTION

[0003] Immune cell proliferation plays a central role in generating potent immune responses, including those of chimeric antigen receptor (CAR)-T cells. Extending cell therapy beyond treatment of blood cancers to solid cancers will require tools to enhance immune cell proliferation locally without systemic side effects.

[0004] There is a need in the art for synthetic immune cells that inducibly secrete proliferative cytokines upon recognition of a local target antigen, such as a tumor antigen.

SUMMARY

[0005] The present disclosure provides a genetically modified, in vitro immune cell. The immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator. The present disclosure provides compositions comprising the genetically modified, in vitro immune cell; and treatment methods comprising administration of the genetically modified, in vitro immune cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1A-1C depict schematically immune cell expansion and balance between amplification and side effects.

[0007] FIG. 2A-2D depict synthetic helper T cells with inducible cytokine circuits for immune cell expansion.

[0008] FIG. 3A-3E depict the effect of synthetic helper T cells on locally targeted proliferation in vivo.

[0009] FIG. 4A-4C depict bioluminescence images of individual mice over 18 days.

[0010] FIG. 5A-5D depict synthetic helpers forming a functional AND-gate circuit with cytotoxic effectors to specifically enhance local killing of target tumors.

[0011] FIG. 6A-6B depict tumor volume trajectories of individual mice.

[0012] FIG. 7 is a schematic depiction of a chimeric Notch receptor polypeptide.

[0013] FIG. 8A-8G provide schematic depictions of exemplary chimeric Notch receptor polypeptides.

[0014] FIG. 9A-9C depict examples of chimeric Notch receptor polypeptides. The sequences are set forth in SEQ ID NOs: 96-98.

[0015] FIG. 10A-10C provide amino acid sequences of exemplary chimeric Notch receptor polypeptides. The sequences are set forth in SEQ ID NOs: 99-101.

[0016] FIG. 11 provides an amino acid sequence of an exemplary chimeric Notch polypeptide. The sequence is set forth in SEQ ID NO: 102.

[0017] FIG. 12A-12B provide amino acid sequences of exemplary chimeric Notch receptor polypeptides. The sequences is set forth in SEQ ID NOs: 103-104.

[0018] FIG. 13A-13D provide amino acid sequences of exemplary chimeric Notch receptor polypeptides. The sequences are set forth in SEQ ID NOs: 105-108.

[0019] FIGS. 14-16 provide amino acid sequences of exemplary chimeric Notch receptor polypeptides. The sequences are set forth in SEQ ID NOs: 109-111.

[0020] FIG. 17A-17C depicts amino acid sequence of IL-2 receptor polypeptides IL-2R.alpha., IL2R.beta., and IL-2R.gamma.. The sequences are set forth in SEQ ID NOs: 112-114.

DEFINITIONS

[0021] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0022] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.

[0023] A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.

[0024] "Heterologous," as used herein, means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively. Heterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acids or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides.

[0025] The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, a single domain light chain antibodies, nanobodies, bi-specific antibodies, multi-specific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. The antibodies can be detectably labeled, e.g., with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. The antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like. The antibodies can also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the term are Fab', Fv, F(ab').sub.2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repetitive cellular replication. That is, the clone of cells only produces a single antibody species. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). An antibody can be monovalent or bivalent. An antibody can be an Ig monomer, which is a "Y-shaped" molecule that consists of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0026] The term "humanized immunoglobulin" as used herein refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion comprises amino acid sequences of human origin. For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising a complementarity-determining region (CDR) derived from an antibody of nonhuman origin and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1. See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

[0027] The term "nanobody" (Nb), as used herein, refers to the smallest antigen binding fragment or single variable domain (V.sub.HH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996). In the family of "camelids" immunoglobulins devoid of light polypeptide chains are found. "Camelids" comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A single variable domain heavy chain antibody is referred to herein as a nanobody or a V.sub.HH antibody.

[0028] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab').sub.2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0029] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and--binding site. This region consists of a dimer of one heavy--and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0030] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH,) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH.sub.1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab').sub.2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0031] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0032] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise the V.sub.H and V.sub.L domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0033] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V.sub.H) connected to a light-chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0034] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (K.sub.D). Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences. Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and/or antigen-binding fragments.

[0035] The term "binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In some cases, a specific binding member present in the extracellular domain of a chimeric polypeptide of the present disclosure binds specifically to its binding partner, such as an antigen or a peptide-major histocompatibility complex (peptide-MHC). "Specific binding" refers to binding with an affinity of at least about 10.sup.-7 M or greater, e.g., 5.times.10.sup.-7 M, 10.sup.-8 M, 5.times.10.sup.-8 M, and greater. "Non-specific binding" refers to binding with an affinity of less than about 10.sup.-7 M, e.g., binding with an affinity of 10.sup.-6 M, 10.sup.-5 M, 10.sup.-4 M, etc.

[0036] The terms "polypeptide," "peptide," and "protein", used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0037] An "isolated" polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some cases, the polypeptide will be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. In some instances, isolated polypeptide will be prepared by at least one purification step.

[0038] The terms "chimeric antigen receptor" and "CAR", used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014/127261 and US Patent Application No. 2015/0368342, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19-4-1BB-CD3.zeta. CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0039] As used herein, the terms "treatment," "treating," "treat" and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0040] A "therapeutically effective amount" or "efficacious amount" refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0041] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit.

[0042] As used herein, the term "immune cells" generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow "Immune cells" includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).

[0043] "T cell" includes all types of immune cells expressing CD3 including T-helper cells (CD4.sup.+ cells), cytotoxic T-cells (CD8.sup.+ cells), T-regulatory cells (Treg) and gamma-delta T cells.

[0044] A "cytotoxic cell" includes CD8.sup.+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.

[0045] The term "synthetic," as used herein in the context of a "synthetic immune cell," generally refers to an artificially derived (e.g., laboratory generated) cell that is not naturally occurring.

[0046] The term "recombinant", as used herein describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell or a virus means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0047] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0048] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0050] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a synthetic immune cell" includes a plurality of such cells and reference to "the IL-2 polypeptide" includes reference to one or more IL-2 polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0051] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0052] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0053] The present disclosure provides a genetically modified, in vitro immune cell. The immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator. The present disclosure provides compositions comprising the genetically modified, in vitro immune cell; and treatment methods comprising administration of the genetically modified, in vitro immune cell.

Genetically Modified Immune Cells

[0054] The present disclosure provides a genetically modified, in vitro immune cell. The immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator. A genetically modified, in vitro immune cell of the present disclosure is also referred to herein as a "synthetic immune cell."

[0055] A genetically modified, in vitro immune cell of the present disclosure can be a T cell or a macrophage. A genetically modified, in vitro immune cell of the present disclosure can be a cytotoxic T cell. A genetically modified, in vitro immune cell of the present disclosure can be a CD8.sup.+ T cell. A genetically modified, in vitro immune cell of the present disclosure can be a CD4.sup.+ T cell.

[0056] A genetically modified, in vitro immune cell of the present disclosure, when administered to an individual in need thereof, provides for an increase in proliferation and/or activity of a second immune cell (a "target immune cell") present in the individual. The second immune cell can be an endogenous immune cell, e.g., an immune cell that is not genetically modified (e.g., not genetically modified ex vivo). For example, the second immune cell can be a T cell that expresses an endogenous T-cell receptor (TCR). The second immune cell can be one that has been genetically modified in vitro and administered to the individual. For example, the second immune cell can be one that has been genetically modified in vitro to express: i) an exogenous TCR; ii) a chimeric antigen receptor (CAR); or iii) a bispecific T-cell engager (BiTE).

[0057] In some cases, a genetically modified, in vitro immune cell of the present disclosure, when administered to an individual in need thereof, provides for an increase in proliferation of a target immune cell in the individual of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the level of proliferation of the target cell before administration of the genetically modified immune cell of the present disclosure.

[0058] In some cases, a genetically modified, in vitro immune cell of the present disclosure, when administered to an individual in need thereof, provides for an increase in cytotoxic T cell activity of a target immune cell in the individual of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold, compared to the cytotoxic activity of the target immune cell before administration of the genetically modified immune cell of the present disclosure.

Cytokines

[0059] As noted above, a genetically modified, in vitro immune cell of the present disclosure is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a cytokine or other proliferation-inducing polypeptide, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator present in the chimeric polypeptide.

[0060] As used herein, the term "cytokine" includes cytokines comprising naturally-occurring ("wild-type") amino acid sequences and cytokines comprising non-naturally-occurring amino acid sequences. For example, a cytokine comprising a non-naturally-occurring amino acid sequence can be referred to as a "variant" cytokine. In some cases, a variant cytokine comprises an amino acid sequence that differs from a naturally-occurring amino acid sequence by from 1 to 25 amino acids, e.g., from 1 amino acid to 5 amino acids, from 5 amino acids to 10 amino acids, from 10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, or from 20 amino acids to 25 amino acids.

Variant IL-2 Polypeptides

[0061] In some cases, a variant IL-2 polypeptide is one that exhibits increased affinity for IL-2 receptor (IL-2R)-beta (IL-2R.beta.). See, e.g., Levin et al. (2012) Nature 484:529; and US 2019/0248860.

[0062] An amino acid sequence of wild-type human IL-2, with the signal peptide (in bold and underlined), is as follows:

TABLE-US-00001 (SEQ ID NO: 1) MYRMQLLSCIALSLALVTNSAPTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT.

[0063] In some cases, the IL-2 lacks the signal sequence. For example, in some cases, the IL-2 comprises the following amino acid sequence:

[0064] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:2).

[0065] In some cases, the IL-2 is a variant IL-2 that comprises L80F, R81D, L85V, I86V, and I92F substitutions, based on the amino acid numbering of SEQ ID NO:2. Thus, e.g., in some cases, the variant IL-2 comprises the following amino acid sequence:

TABLE-US-00002 (SEQ ID NO: 4) MYRMQLLSCIALSLALVTNSAPTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHF DPRDVVISNI NVFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT.

[0066] In some cases, the IL-2 is a variant IL-2 that comprises L80F, R81D, L85V, I86V, and I92F substitutions, based on the amino acid numbering of SEQ ID NO:2; and lacks a signal sequence. For example, in some cases, the variant IL-2 comprises the following amino acid sequence:

[0067] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHF DPRDVVISNIN VFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:5); and has a length of 133 amino acids. Such a variant IL-2 is also referred to as "super 2." Such a variant IL-2 exhibits higher affinity for IL-2R.beta. than an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. For example, such a variant IL-2 exhibits an affinity for IL-2R.beta. that is at least 20%, at least 25%, at least 50%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, or at least 10-fold, higher than the affinity of the IL-2 polypeptide of SEQ ID NO:2 for IL-2R.beta..

[0068] In some cases, the variant IL-2 includes, in addition to L80F, R81D, L85V, I86V, and I92F substitutions, an amino acid substitution at one or more of I24, F42, K43, P65, Q74, I89, and V93, based on the amino acid numbering of SEQ ID NO:2. In some cases, the variant IL-2 further includes a K43N amino acid substitution, based on the amino acid numbering of SEQ ID NO:2. In some cases, the variant IL-2 includes, in addition to L80F, R81D, L85V, I86V, and I92F substitutions, one or more amino acid substitutions selected from I24V, P65H, Q74R, Q74H, Q74N, Q74S, I89V, and V93I, based on the amino acid numbering of SEQ ID NO:2.

[0069] In some cases, the variant IL-2 comprises the following amino acid substitutions: F42A, L80F, R81D, L85V, I86V, I89V, and I92F, based on the amino acid numbering of SEQ ID NO:2. Thus, for example, in some cases, the variant IL-2 comprises the following amino acid sequence: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHF DPRDVVISNVN VFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:6).

[0070] In some cases, the variant IL-2 comprises the following amino acid substitutions: L80F, R81D, L85V, I86V, I89V, 192F, and V93I, based on the amino acid numbering of SEQ ID NO:2. Thus, for example, in some cases, the variant IL-2 comprises the following amino acid sequence: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHF DPRDVVISNVN VFILELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:7).

[0071] In some cases, the variant IL-2 comprises the following amino acid substitutions: Q74H, L80F, R81D, L85V, I86V, and 192F, based on the amino acid numbering of SEQ ID NO:2. Thus, for example, in some cases, the variant IL-2 comprises the following amino acid sequence: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAHSKNFHF DPRDVVISNIN VFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:8).

[0072] In some cases, the variant IL-2 comprises the following amino acid substitutions: Q74S, L80F, R81D, L85V, I86V, and I92F, based on the amino acid numbering of SEQ ID NO:2. Thus, for example, in some cases, the variant IL-2 comprises the following amino acid sequence: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLASSKNFHF DPRDVVISNIN VFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:9).

[0073] In some cases, the variant IL-2 comprises the following amino acid substitutions: Q74N, L80F, R81D, L85V, I86V, and 192F, based on the amino acid numbering of SEQ ID NO:2. Thus, for example, in some cases, the variant IL-2 comprises the following amino acid sequence: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLANSKNFHF DPRDVVISNIN VFVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:10).

Orthogonal IL-2 Variant That Binds IL-2R With Variant IL-2R.beta.

[0074] A suitable IL-2 variant is one that binds selectively to an IL-2R comprising a variant IL-2R.beta.; such a variant IL-2 is referred to as "ortho IL-2."

[0075] An amino acid sequence of wild-type human IL-2, with the signal peptide (in bold and underlined), is as follows:

TABLE-US-00003 (SEQ ID NO: 1) MYRMQLLSCIALSLALVTNSAPTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT.

[0076] In some cases, the IL-2 lacks the signal sequence. For example, in some cases, wild-type human IL-2 comprises the following amino acid sequence:

TABLE-US-00004 (SEQ ID NO: 2) APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT.

[0077] Wild-type human IL-2 binds to an IL-2 receptor (IL-2R) on the surface of a cell. An IL-2 receptor is in some cases a heterotrimeric polypeptide comprising an alpha chain (IL-2R.alpha.; also referred to as CD25), a beta chain (IL-2R.beta.; also referred to as CD122: and a gamma chain (IL-2R.gamma.; also referred to as CD132) Amino acid sequences of human IL-2R.alpha., human IL-2R.beta., and human IL-2R.gamma. are provided in FIG. 17A-17C.

[0078] An ortho-IL-2 polypeptide can comprise amino acid substitutions at one or more amino acids selected from Q13, L14, E15, H16, L19, D20, Q22, M23, G27, and N88, based on the amino acid numbering of SEQ ID NO:2. An ortho-IL-2 polypeptide can comprise amino acid substitutions at one or more amino acids selected from E15, H16, L19, D20, Q22, and M23, based on the amino acid numbering of SEQ ID NO:2. An ortho-IL-2 polypeptide can comprise one or more of the following amino acid substitutions: Q13W, L14M, L14W, E15D, E15T, E15A, E155, H16N, H16Q, L19V, L19I, L19A, D20L, D20M, Q22S, Q22T, Q22E, Q22K, Q22E, M23A, M23W, M23H, M23Y, M23F, M23Q, M23Y, G27K, G27S, R81D, R81Y, N88E, N88Q, and T51I. An ortho-IL-2 polypeptide can comprise H16N, L19V, D20N, Q22T, M23H, and G27K substitutions. An ortho-IL-2 polypeptide can comprise E15D, H16N, L19V, D20L, Q22T, and M23H substitutions. An ortho-IL-2 polypeptide can comprise E15D, H16N, L19V, D20L, Q22T, and M23A substitutions. An ortho-IL-2 polypeptide can comprise E15D, H16N, L19V, D20L, Q22K, M23A substitutions.

[0079] In some cases, an ortho-IL-2 polypeptide comprises one of the following sets of substitutions: [E15S; H16Q; L19V, D20T/S; Q22K, M23L/S]; [E15S; H16Q; L19I; D20S; Q22K; M23L]; [E15S; L19V; D20M; Q22K; M23S]; [E15T; H16Q; L19V; D20S; M23S]; [E15Q; L19V; D20M; Q22K; M23S]; [E15Q; H16Q; L19V; D20T; Q22K; M23V]; [E15H; H16Q; L19I; D20S; Q22K; M23L]; [E15H; H16Q; L19I; D20L; Q22K; M23T]; [19V; D20M; Q22N; M23S]; [E15S, H16Q, L19V, D20L, M23Q, R81D, T51I], [E15S, H16Q, L19V, D20L, M23Q, R81Y], [E15S, H16Q, L19V, D20L, Q22K, M23A], [E15S, H16Q, L19V, D20L, M23A].

[0080] In some cases, an ortho-IL-2 polypeptide comprises E15S5, H16Q, L19V, D20T/S/M, Q22K, and M23L/S substitutions, based on the amino acid numbering of SEQ ID NO:2. In some cases, an ortho-IL-2 polypeptide comprises E15S, H16Q, L19V, D20L, and M23Q/A substitutions, based on the amino acid numbering of SEQ ID NO:2. For example, in some cases, an ortho-IL-2 polypeptide comprises the amino acid sequence: APTSSSTKKT QLQLSQLLVT LKLILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:11).

[0081] Wild-type human IL-2R.beta. (mature; without signal peptide) can have the following amino acid sequence:

TABLE-US-00005 (SEQ ID NO: 3) AVNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEV PDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V

[0082] In some cases, a variant IL-2R.beta. (a variant to which an ortho-IL-2 polypeptide binds) comprises an amino acid substitution at one or more amino acids selected from R41, R42, Q70, K71, T73, T74, V75, S132, H133, Y134, F135, E136, and Q214, based on the amino acid numbering of SEQ ID NO:3. In some cases, a variant IL-2R.beta. (a variant to which an ortho-IL-2 polypeptide binds) comprises an amino acid substitution at one or more amino acids selected from Q70, T73, H133, and Y134, based on the amino acid numbering of SEQ ID NO:3.

[0083] In some cases, a variant IL-2R.beta. (a variant to which an ortho-IL-2 polypeptide binds) comprises one or more amino acid substitutions selected from Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R, based on the amino acid numbering of SEQ ID NO:3.

[0084] As one example, a variant IL-2R.beta. (a variant to which an ortho-IL-2 polypeptide binds) has the following amino acid sequence (with Q70Y, T73D, H133D, and Y134F substitutions, shown in bold, compared to SEQ ID NO:3):

TABLE-US-00006 (SEQ ID NO: 12) AVNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSYKLDT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI SWEISQASDF FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEV PDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V.

Chimeric Polypeptide

[0085] As noted above, a genetically modified, in vitro immune cell of the present disclosure is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator. In some cases, the chimeric polypeptide is a chimeric Notch polypeptide ("synNotch"). Chimeric Notch polypeptides are described in, e.g., WO 2016/138034; and U.S. Pat. No. 9,670,281. In some cases the chimeric polypeptide is a force sensor cleavage domain-containing chimeric polypeptide (also referred to herein as an "A2 chimeric polypeptide"). Force sensor cleavage domain-containing chimeric polypeptides are described in, e.g., WO 2019/099689.

Chimeric Notch Polypeptides

[0086] In some cases, the chimeric polypeptide is a chimeric Notch polypeptide (a "synNotch" polypeptide). In some cases, the chimeric polypeptide comprises, from N-terminal to C-terminal and in covalent linkage: i) an extracellular domain comprising an antibody specific for a target antigen; ii) a Notch regulatory polypeptide (also referred to as a "Notch receptor polypeptide") that comprises one or more proteolytic cleavage sites; and; iii) an intracellular domain comprising a transcriptional activator. Binding of the antibody to the target antigen (e.g., a target antigen present on the surface of a cell) induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain.

[0087] In some cases, the Notch regulatory polypeptide has a length of from 300 amino acids to 400 amino acids. In some cases, the one or more binding-inducible proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a .gamma.-secretase cleavage site comprising a Gly-Val dipeptide sequence. In some cases, the Notch regulatory polypeptide comprises a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site. In some cases, the Notch regulatory polypeptide further comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats. In some cases, the chimeric polypeptide comprises a linker interposed between the extracellular domain and the Notch regulatory polypeptide. In some cases, the target antigen is a cancer-associated antigen (e.g., a tumor-specific antigen), a disease-associated antigen, a pathogen-associated antigen, an autoimmune disease-associated antigen, or an extracellular matrix component. In some cases, the antibody is a single-chain Fv. In some cases, the antibody is a nanobody, a single-domain antibody, a diabody, a triabody, or a minibody.

Extracellular Domain

[0088] As noted above, a chimeric Notch receptor polypeptide comprises an extracellular domain comprising an antibody specific for a target antigen. In some cases, the target antigen is present on the surface of a target cell.

[0089] The antibody can be any antigen-binding antibody-based polypeptide, a wide variety of which are known in the art. In some instances, the antibody is a single chain Fv (scFv). Other antibody based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and "camelized" antibody variable domains are suitable for use. In some cases, the antibody is a nanobody, a single-domain antibody, a diabody, a triabody, or a minibody.

[0090] In some cases, the antigen-binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a cancer cell, i.e., a cancer cell associated antigen. The cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancer cell associated antigen may also be expressed by a non-cancerous cell.

[0091] In some cases, the antigen-binding domain is specific for an epitope present in a tissue-specific antigen. In some cases, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0092] Non-limiting examples of antigens to which an antigen-binding domain of a subject chimeric Notch receptor polypeptide can bind include, e.g., CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

[0093] Non-limiting examples of antigens to which an antigen-binding domain of a subject chimeric Notch receptor polypeptide can bind include, e.g., Cadherins (CDH1-20), Integrins (alfa and beta isoforms), Ephrins, NCAMs, connexins, CD44, syndecan, CD47, DGalfa/beta, SV2, protocadherin, Fas, Dectin-1, CD7, CD40, Neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4, CD28, TIGIT, and ICOS.

[0094] In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell-surface receptor.

[0095] In some cases, the antibody is specific for a cell surface target, where non-limiting examples of cell surface targets include CD19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD 11a, and alpha-integrin.

[0096] Where the antibody is specific for a cancer-associated antigen, the antigen can be a cancer-associated antigen, where cancer-associated antigens include, e.g., CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Cancer-associated antigens also include, e.g., 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin .alpha.5.beta.1, integrin .alpha.v.beta.3, MORAb-009, MS4A1, MUCl, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R .alpha., PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-.beta., TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0097] The antigen can be associated with an inflammatory disease. Non-limiting examples of antigens associated with inflammatory disease include, e.g., AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (.alpha.chain of IL-2 receptor), CD3, CD4, CDS, IFN-.alpha., IFN-.gamma., IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin .alpha.4, integrin .alpha.4.beta.7, LFA-1 (CD11.alpha.), myostatin, OX-40, scleroscin, SOST, TGF beta 1, TNF-.alpha., and VEGF-A.

Notch Regulatory Polypeptide

[0098] In some cases, the Notch receptor polypeptide (also referred to herein as a "Notch regulatory polypeptide") present in a chimeric Notch receptor polypeptide has a length of from 50 amino acids (aa) to 1000 aa, e.g., from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 a to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa, from 750 aa to 800 aa, from 800 aa to 850 aa, from 850 aa to 900 aa, from 900 aa to 950 aa, or from 950 aa to 1000 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 300 aa to 400 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 300 aa to 350 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 300 aa to 325 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 350 aa to 400 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 750 aa to 850 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of from 50 aa to 75 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 310 aa to 320 aa, e.g., 310 aa, 311 aa, 312 aa, 313 aa, 314 aa, 315 aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of 315 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of from 360 aa to 370 aa, e.g., 360 aa, 361 aa, 362 aa, 363 aa 364 aa, 365 aa, 366 aa, 367 aa, 368 aa, 369 aa, or 370 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide has a length of 367 aa.

[0099] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

TABLE-US-00007 (SEQ ID NO: 13) IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR QLCIQKL;

where the transmembrane.TM. domain is underlined; where the Notch receptor polypeptide comprises an S2 proteolytic cleavage site and an S3 proteolytic cleavage site; where the Notch receptor polypeptide has a length of from 50 amino acids (aa) to 65 aa, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

TABLE-US-00008 (SEQ ID NO: 13) IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR QLCIQKL;

where the TM domain is underlined; where the Notch receptor polypeptide comprises an S2 proteolytic cleavage site and an S3 proteolytic cleavage site; where the Notch receptor polypeptide has a length of 56 amino acids.

[0100] In some cases, the Notch regulatory polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) a LNR-A segment; ii) a LNR-B segment; iii) a LNR-C segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain A LNR-A segment, LNR-B segment, and LNR-C segment can collectively be referred to as an "LNR segment."

[0101] An LNR segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDG HCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDC (SEQ ID NO:15); and can have a length of from 118 to 122 amino acids (e.g., 118, 119, 120, 121, or 122 amino acids).

[0102] An HD segment (HD-N plus HD-C) can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKR STVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALAS LGSLNIPYKIEAVKSEPVEPPLP (SEQ ID NO:16); and can have a length of 150, 151, 152, 153, or 154 amino acids.

[0103] A transmembrane segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:17); and can have a length of 21, 22, 23, 24, or 25 amino acids.

[0104] In some cases, the Notch regulatory polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the following amino acid sequence:

TABLE-US-00009 (SEQ ID NO: 18) PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQ SLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDG HCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLR ELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLP GTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGAL ASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLL S.

[0105] In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to the following sequence:

PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQ VDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLF DGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVV LLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSS LLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKI EAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:19). In some cases, the one or more ligand-inducible proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. In some cases, the 51 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a .gamma.-secretase cleavage site comprising a Gly-Val dipeptide sequence.

[0106] A schematic depiction of a Notch receptor polypeptide is provided in FIG. 7. The Notch receptor polypeptide depicted in FIG. 7 includes: a) an extracellular portion that includes: i) epidermal growth factor (EGF) repeats; ii) a ligand binding site; iii) three Lin-12 Notch repeats (LNR), designated LNR-A, LNR-B, and LNR-C; iv) two heterodimerization domains (HD-N and HD-C); b) a transmembrane (TM) portion; and c) an intracellular portion that includes: i) a RAM domain; ii) ankyrin repeats; iii) a transcription activation domain; and iv) a PEST region. A Notch receptor polypeptide includes three proteolytic sites, termed 51, S2, and S3. 51, a furin cleavage site, is located between HD-N and HC-C; S2, an ADAM17 cleavage site, is located within HD-C; and S3, a gamma secretase cleavage site, is within the TM portion. A Notch receptor polypeptide mediates cell-to-cell communication, e.g. communication between contacting cells, in which one contacting cell is a "receiver" cell and the other contacting cell is a "sender" cell. Engagement of a Notch receptor polypeptide present on a receiving cell by a Delta polypeptide ("ligand") present on a sending cell results in ligand-induced cleavage of the Notch receptor polypeptide, resulting in release of the intracellular portion of the receptor from the membrane into the cytoplasm. The released portion alters receiver cell behavior by functioning as a transcriptional regulator.

[0107] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) a single EGF repeat; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain

[0108] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) a LNR-A segment; ii) a LNR-B segment; iii) a LNR-C segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain An LNR-A segment, LNR-B segment, and LNR-C segment can collectively be referred to as an "LNR segment." Such a Notch receptor polypeptide is depicted schematically in FIG. 8A.

[0109] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) a single EGF repeat; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain Such a Notch receptor polypeptide is depicted schematically in FIG. 8B

[0110] In some cases, a Notch receptor polypeptide comprises a synthetic linker. For example, in some cases, a Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: i) a synthetic linker; ii) an EGF repeat; iii) an LNR segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain Such a Notch receptor polypeptide is depicted schematically in FIG. 8C.

[0111] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) from two to eleven EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain Such a Notch receptor polypeptide is depicted schematically in FIG. 8D.

[0112] In some cases, a Notch receptor polypeptide comprises a synthetic linker. For example, in some cases, a Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: i) two to eleven EGF repeats; ii) a synthetic linker; iii) an LNR segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain. Such a Notch receptor polypeptide is depicted schematically in FIG. 8E.

[0113] A synthetic linker can have a length of from about 10 amino acids (aa) to about 200 aa, e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 125 aa, from 125 aa to 150 aa, from 150 aa to 175 aa, or from 175 aa to 200 aa. A synthetic linker can have a length of from 10 aa to 30 aa, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aa. A synthetic linker can have a length of from 30 aa to 50 aa, e.g., from 30 aa to 35 aa, from 35 aa to 40 aa, from 40 aa to 45 aa, or from 45 aa to 50 aa.

[0114] In some cases, a Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: i) an HD-C segment; and ii) a TM domain, where the Notch receptor polypeptide does not include an LNR segment. In some cases, the LNR segment is replaced with a heterologous polypeptide. Such a Notch receptor polypeptide is depicted schematically in FIG. 8F.

[0115] In some cases, the Notch receptor polypeptide lacks an S1 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S3 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide includes an S3 ligand-inducible proteolytic cleavage site; and lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. Examples are depicted schematically in FIG. 8G.

[0116] An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following sequence: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH (SEQ ID NO:20); and can have a length of 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids. An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence DINECVLSPCRHGASCQNTHGGYRCHCQAGYSGRNCE (SEQ ID NO:21); and can have a length of from 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa). An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence DIDDCRPNPCHNGGSCTDGINTAFCDCLPGFRGTFCE (SEQ ID NO:22); and can have a length of from 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa). Other suitable EGF repeat sequences include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to: i) DVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQ (SEQ ID NO:23) (where the EGF repeat has a length of from 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa)); ii) LVDECSPSPCQNGATCTDYLGGYSCKCVAGYHGVNC (SEQ ID NO:24) (where the EGF repeat has a length of from 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa)); iii) IDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHCE (SEQ ID NO:25) (where the EGF repeat has a length of from 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa)); and iv) CFNNGTCVDQVGGYSCTCPPGFVGERC (SEQ ID NO:26) (where the EGF repeat has a length of from 25 amino acids (aa) to 30 aa, e.g., 25, 26, 27, 28, 29, or 30 aa).

[0117] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) two EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) three EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) four EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) five EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) six EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) seven EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) eight EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) nine EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) ten EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: i) eleven EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain

[0118] In some cases, the Notch receptor polypeptide includes only one binding-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide includes two binding-inducible proteolytic cleavage sites. In some cases, the Notch receptor polypeptide includes three binding-inducible proteolytic cleavage sites. For simplicity, binding-inducible cleavage sites will be referred to herein as "S1," "S2," and "S3" binding-inducible proteolytic cleavage sites.

[0119] In some cases, the Notch receptor polypeptide includes an S1 ligand-inducible proteolytic cleavage site. An S1 ligand-inducible proteolytic cleavage site can be located between the HD-N segment and the HD-C segment. In some cases, the S1 ligand-inducible proteolytic cleavage site is a furin-like protease cleavage site. A furin-like protease cleavage site can have the canonical sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid; the protease cleaves immediately C-terminal to the canonical sequence. For example, in some cases, an amino acid sequence comprising an S1 ligand-inducible proteolytic cleavage site can have the amino acid sequence GRRRRELDPM (SEQ ID NO:27), where cleavage occurs between the "RE" sequence. As another example, an amino acid sequence comprising an S1 ligand-inducible proteolytic cleavage site can have the amino acid sequence RQRRELDPM (SEQ ID NO:28), where cleavage occurs between the "RE" sequence.

[0120] In some cases, the Notch receptor polypeptide includes an S2 ligand-inducible proteolytic cleavage site. An S2 ligand-inducible proteolytic cleavage site can be located within the HD-C segment. In some cases, the S2 ligand-inducible proteolytic cleavage site is an ADAM-17-type protease cleavage site. An ADAM-17-type protease cleavage site can comprise an Ala-Val dipeptide sequence, where the enzyme cleaves between the Ala and the Val. For example, in some cases, amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVKSE (SEQ ID NO:29), where cleavage occurs between the "AV" sequence. As another example, an amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVQSE (SEQ ID NO:30), where cleavage occurs between the "AV" sequence.

[0121] In some cases, the Notch receptor polypeptide includes an S3 ligand-inducible proteolytic cleavage site. An S3 ligand-inducible proteolytic cleavage site can be located within the TM domain In some cases, the S3 ligand-inducible proteolytic cleavage site is a gamma-secretase (.gamma.-secretase) cleavage site. A y-secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between the Gly and the Val. For example, in some cases, an S3 ligand-inducible proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO:31), where cleavage occurs between the "GV" sequence. In some cases, an S3 ligand-inducible proteolytic cleavage site comprises the amino acid sequence GCGVLLS (SEQ ID NO:32).

[0122] In some cases, the Notch receptor polypeptide lacks an S1 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S3 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide includes an S3 ligand-inducible proteolytic cleavage site; and lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site.

Intracellular Domain

[0123] A chimeric Notch polypeptide comprises an intracellular domain comprising a binding triggered transcriptional activator. Binding of the antibody to its target antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain. The transcriptional activator binds to a transcriptional control element that is operably linked to the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide, and activates transcription, such that the cytokine or proliferation-inducing polypeptide is produced.

[0124] Non-limiting examples of suitable transcriptional regulators include, e.g., ABT1, ACYP2, AEBP1, AEBP2, AES, AFF1, AFF3, AHR, ANK1, ANK2, ANKFY1, ANKIB1, ANKRD1, ANKRD10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B, AR, ARHGAP17, ARID1A, ARID1B, ARID3A, ARID4A, ARID5B, ARNT, ARNT2, ARNTL, ARNTL2, ARX, ASB10, ASB11, ASB12, ASB15, ASB2, ASB5, ASB8, ASB9, ASH1L, ASH2L, ASXL1, ASZ1, ATF1, ATF3, ATF4, ATF4, ATF5, ATF6, ATF7, ATF7IP, ATM, ATOH1, ATXN3, 1300003B13RIK, B3GAT3, B930041F14RIK, BACH1, BACH2, BARX1, BARX2, BATF, BATF2, BATF3, BAZ2A, BBX, BC003267, BCL11A, BCL11B, BCL3, BCL6, BCL6B, BCLAF1, BCOR, BHLHA15, BHLHE40, BHLHE41, BLZFl, BMYC, BNC1, BNC2, BPNT1, BRCA1, BRWD1, BTBD11, BTF3, 6030408C04RIK, CAMK4, CARHSP1, CARM1, CBX4, CBX7, CCNC, CCNH, CCNT1, CCNT2, CDC5L, CDK2, CDK4, CDK9, CDKN2C, CDX1, CDX1, CDX2, CEBPA, CEBPB, CEBPD, CEBPG, CEBPG, CEBPZ, CHD4, CHD7, CHGB, CIC, CIITA, CITED1, CITED2, CITED4, CLOCK, CLPB, CML3, CNOT7, COPS2, CREB1, CREB3, CREB3L1, CREB3L1, CREB3L2, CREB3L3, CREB5, CREBBP, CREBL2, CREM, CSDA, CSDA, CSDC2, CSDE1, CTBP2, CTCF, CTCFL, CTNNB1, CTNNBL1, CXXCl, D11BWG0517E, 2300002D11RIK, DACH1, DAXX, DBP, DDIT3, DDX20, DDX54, DDX58, DEAF1, DEK, DIDO1, DLX2, DMRT1, DMRT2, DMRTB1, DNMT1, DNMT3A, DR1, DRG1, DUSP26, DYSFIP1, E2F1, E2F2, E2F3, E2F5, E2F6, EBF1, EBF2, EBF3, EBF3, EED, EGR1, EGR2, EGR3, EHF, EHMT2, EID2, ELAVL2,ELF1, ELF1, ELF2, ELF3, ELF4, ELF5, ELK3, ELK4, ELL2, EMX2, EMX2, EN2, ENPP2, EOMES, EP300, EPAS1, ERF, ERG, ESR1, ESRRA, ESRRB, ESRRG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV5, ETV6, EVI1, EWSR1, EZH1, EZH2, FAH, FBXL10, FBXL11, FBXW7, FEM1A, FEM1B, FEM1C, FHL2, FLI1, FMNL2, FOS, FOSB, FOSL1, FOSL2, FOXA1, FOXA2, FOXA3, FOXCl, FOXD1, FOXD2, FOXD3, FOXF1, FOXF1A, FOXF2, FOXG1, FOXI1, FOXJ2, FOXJ3, FOXKl, FOXK2, FOXL1, FOXL2, FOXMl, FOXN1, FOXN2, FOXN3, FOXO1, FOXO3, FOXP1, FOXP2, FOXP3, FOXP4, FOXQ1, FUS, FUSIP1, 2810021G02RIK, GABPA, GABPB1, GARNL1, GAS7, GATA1, GATA2, GATA3, GATA4, GATA5, GATA5, GATA6, GBX2, GCDH, GCM1, GFIl, GFI1B, GLI2, GLI3, GLIS1, GLIS2, GLIS3, GLS2, GMEB1, GMEB2, GRHL1, GRHL2, GRHL3, GRLF1, GTF2A1, GTF2B, GTF2E2, GTF2F1, GTF2F2, GTF2H2, GTF2H4, GTF2I, GTF2IRD1, GTF2IRD1, GZFl, HAND2, HBP1, HCLS1, HDAC10, HDAC11, HDAC2, HDAC5, HDAC9, HELZ, HEST, HES4, HESS, HES6, HEXIM1, HEY2, HEYL, HHEX, HHEX, HIC1, HIC2, HIF1A, HIF1AN, HIPK2, HIVEP1, HIVEP2, HIVEP2, HIVEP3, HLF, HLTF, HLX, HMBOX1, HMG20A, HMGA2, HMGB2, HMGB3, HNF1B, HNF4A, HNF4G, HOMEZ, HOXA10, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9, HOXC10, HOXC10, HOXC11, HOXC5, HOXC6, HOXC8, HOXC9, HOXD8, HOXD9, HR, HSBP1, HSF2BP, HTATIP2, HTATSF1, HUWE1, 5830417I10RIK, ID1, ID2, ID3, ID3, IFNAR2, IKBKB, IKBKG, IKZFl, IKZF2, IKZF3, IKZF4, IL31RA, ILF3, ING1, ING2, ING3, ING4, INSM1, INTS12, IQWD1, IRF1, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF8, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, ISL2, ISX, ISX, IVNS1ABP, 2810021J22RIK, JARID1A, JARID1B, JARID1C, JARID1D, JDP2, JUN, JUNB, JUND, KLF1, KLF10, KLF11, KLF12, KLF13, KLF15, KLF16, KLF2, KLF3, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KRR1, 6330416L07RIK, L3MBTL2, LASS2, LASS4, LASS6, LBA1, LBH, LBX1, LCOR, LDB1, LDB2, LEFT, LHX1, LHX2, LHX5, LIMD1, LIN28, LMO1, LMO4, LMX1A, LSM11, LSM4, LYL1, 9030612M13RIK, 1810007M14RIK, 3632451006RIK, MAF, MAFA, MAFB, MAFF, MAFG, MAFK, MAGED1, MAP3K12, MAPK1, MAPK3, MAPK8, MAPK8IP1, MAX, MAZ, MBD2, MCM2, MCM4, MCM5, MCM6, MCMI, MECOM, MECP2, MED12, MEDS, MEF2A, MEF2B, MEF2C, MEF2D, MEIS1, MEIS1, MEIS2, MEOX2, MESP2, MIDI, MITF, MKI67IP, MKL1, MLL1, MLL3, MLLT10, MLLT3, MLX, MLXIP, MLXIPL, MNT, MNX1, MPL, MSC, MSRB2, MSX2, MTA3, MTF1, MTF2, MTPN, MXD1, MXD4, MXI1, MYB, MYBBP1A, MYBL2, MYC, MYCBP, MYCL1, MYCN, MYEF2, MYF6, MYNN, MYOCD, MYOD1, MYOG, MYST3, MYST4, MYT1L, MZFl, NAB1, NAB2, NANOG, NARG1, NCOA1, NCOA2, NCOA3, NCOR1, NCOR2, NDN, NEUROD1, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NFAT5, NFATC1, NFATC2, NFATC2IP, NFATC3, NFATC3, NFATC4, NFE2, NFE2L1, NFE2L2, NFIA, NFIA, NFIB, NFIC, NFIL3, NFIX, NFKB1, NFKB2, NFKBIB, NFKBIE, NFKBIZ, NFX1, NFXL1, NFYA, NFYB, NHLH1, NKX2-2, NKX2-3, NKX2-5, NKX2-6, NKX6-2, NMI, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NPAS1, NPAS2, NPAS3, NROB1, NROB2, NR1D1, NR1D2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A2, NR4A3, NR5A1, NR5A2, NRARP, NRIP1, NRIP2, NSBP1, NSD1, NUDT12, NULL, NUPR1, 1700065013RIK, OLIG1, OLIG2, OLIG2, ONECUT1, ONECUT2, ONECUT3, ORC2L, OSGIN1, OSR1, OSR2, OSTF1, OVOL1, OVOL2, PAPOLA, PAPOLG, PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7, PAX8, PAX9, PBX1, PBX2, PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1, PEG3, PERI, PFDN1, PGR, PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A, PHF5A, PHF7, PHOX2A, PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1, PKNOX2, PLA2G6, PLAGL1, PLAGL2, PLRG1, PML, POGK, POLR2B, POLR2E, POLR2H, POLR3E, POLR3H, POLRMT, POU1F1, POU2AF1, POU2F1, POU2F2, POU3F2, POU3F3, POU3F3, POU5F1, POU6F1, PPARA, PPARD, PPARG, PPARGC1A, PPARGC1B, PPP1R12C, PPP1R13B, PPP1R16B, PPP1R1B, PPP2R1A, PPP3CB,PQBP1, PRDM1, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM8, PREB, PRKAR1A, PRKCBP1, PROX1, PRRX1, PRRX2, PSMC5, PSMD10, PSMD9, PTF1A, PTGES2, PURB, PWP1, RAB11A, RAB11B, RAB15, RAB18, RAB1B, RAB25, RAB8A, RAB8B, RAI14, RARA, RARB, RARG, RASSF7, RB1, RBBP7, RBL1, RBM14, RBM39, RBM9, RBPJ, RBPJL, RCOR2, REL, RELA, RELB, RERE, REST, REXO4, RFC1, RFX1, RFX2, RFX3, RFX5, RFX7, RFX8, RHOX5, RHOX6, RHOX9, RIPK4, RNF12, RNF14, RNF141, RNF38, RNF4, RORA, RORA, RORB, RORC, RPS6KA4, RREB1, RSRC1, RUNX1, RUNX1T1, RUNX2, RUNX2, RUNX3, RUVBL1, RUVBL2, RXRA, RXRG, RYBP, SAFB2, SALL1, SALL1, SALL2, SALL4, SAP30, SAP3OBP, SATB1, SATB2, SATB2, SCAND1, SCAP, SCRT2, SEC14L2, SERTAD1, SF1, SFPI1, SFRS5, SH3D19, SH3PXD2B, SHANK3, SHOX2, SHPRH, SIN3A, SIN3B, SIRT2, SIRT3, SIRT5, SIX1, SIX1, SIX2, SIX3, SIX4, SIX5, SKI, SMAD1, SMAD2, SMAD3,SMAD7, SMARCA1, SMARCA2, SMARCA5, SMARCB1, SMYD1, SNAIl, SNAI2, SNAPC2, SNAPC4, SNIP1, SOLH, SOX1, SOX10, SOX11, SOX12, SOX13, SOX15, SOX17, SOX18, SOX2, SOX21, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SP1, SP110, SP140L, SP2, SP3, SP4, SP6, SP8, SPDEF, SPEN, SPI1, SPIB, SQSTM1, SREBF1, SREBF2, SREBF2, SRF, SSBP2, SSBP3, SSBP4, SSRP1, ST18, STAG1, STAT1, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT5B, STATE, SUB1, SUZ12, TADA2L, TAF13, TAF5, TAF5L, TAF7, TAF9, TAL1,TAL1, TARDBP, TBPL1, TBR1, TBX1, TBX10, TBX15, TBX18, TBX2, TBX2, TBX20, TBX21, TBX3, TBX4, TBX5, TBX6, TCEA1, TCEA3, TCEAL1, TCEB3, TCERG1, TCF12, TCF15, TCF19, TCF20, TCF21, TCF21, TCF3, TCF4, TCF7, TCF7L2, TCFAP2A, TCFAP2B, TCFAP2C, TCFCP2L1, TCFE2A, TCFE3, TCFEB, TCFEC, TCFL5, TEAD1, TEAD2, TEAD3, TEAD4, TEF, TFAP2A, TFAP2C, TFCP2L1, TFDP2, TFEB, TFEC, TGFB1I1, TGIF1, TGIF2, TGIF2LX, THRA, THRAP3, THRB, THRSP, TIAL1, TLE1, TLE6, TMEM131, TMPO, TNFAIP3, TOB1, TOX4, TP63, TRERF1, TRIB3, TRIM24, TRIM28, TRIM30, TRIP13, TRIP4, TRIPE, TRP53, TRP53BP1, TRP63, TRPS1, TRPS1, TSC22D1, TSC22D2, TSC22D3, TSC22D4, TSHZ1, TSHZ1, TSHZ3, TTRAP, TUB, TULP4, TWIST1, TWIST2, TYSND1, UBE2W, UBN1, UBP1, UBTF, UGP2, UHRF1, UHRF2, UNCX, USF1, USF2, UTF1, VDR, VEZFl, VGLL2, VSX1, WASL, WHSC1, WHSC2, WT1, WWP1, WWTR1, XBP1, YAF2, YY1, ZBED1, ZBED4, ZBTB1, ZBTB10, ZBTB16, ZBTB16, ZBTB17, ZBTB2, ZBTB20, ZBTB22, ZBTB25, ZBTB32, ZBTB38, ZBTB4, ZBTB43, ZBTB45, ZBTB47, ZBTB7A, ZBTB7B, ZBTB7C, ZCCHC8, ZDHHC13, ZDHHC16, ZDHHC21, ZDHHC5, ZDHHC6, ZEB2, ANK2ZEB2, ZFHX2, ZFHX3, ZFHX4, ZFP105, ZFP110, ZFP143, ZFP148, ZFP161, ZFP192, ZFP207, ZFP219, ZFP238, ZFP263, ZFP275, ZFP277, ZFP281, ZFP287, ZFP292, ZFP35, ZFP354C, ZFP36, ZFP36L1, ZFP386, ZFP407, ZFP42, ZFP423, ZFP426,ZFP445, ZFP451, ATF5ZFP451, ZFP467, ZFP52, ZFP57, ZFP592, ZFP593, ZFP597, ZFP612, ZFP637, ZFP64, ZFP647, ZFP748, ZFP810, ZFP9, ZFP91, ZFPM1, ZFPM2, ZFX, ZHX2, ZHX3, ZIC1, ZIC2, ZIC3, ZIC4, ZIC5, ZKSCAN1, ZKSCAN3, ZMYND11, ZNF143, ZNF160, ZNF175, ZNF184, ZNF192, ZNF213, ZNF217, ZNF219, ZNF22, ZNF238, ZNF24, ZNF267, ZNF273, ZNF276, ZNF280D, ZNF281, ZNF292, ZNF311, ZNF331, ZNF335, ZNF337, ZNF33B, ZNF366, ZNF394, ZNF398, ZNF41, ZNF410, ZNF415, ZNF423, ZNF436, ZNF444, ZNF445, ZNF451, ZNF460, ZNF496, ZNF498, ZNF516, ZNF521, ZNF532, ZNF536, ZNF546, ZNF552, ZNF563, ZNF576, ZNF580, ZNF596, ZNF621, ZNF628, ZNF648, ZNF649, ZNF652, ZNF655, ZNF664, ZNF668, ZNF687, ZNF692, ZNF696, ZNF697, ZNF710, ZNF80, ZNF91, ZNF92, ZNRD1, ZSCAN10, ZSCAN16, ZSCAN20, ZSCAN21, ZXDC, and ZZZ3.

[0125] In some cases, the intracellular domain is a transcription factor. Suitable transcription factors include, e.g., ASCL1, BRN2, CDX2, CDX4, CTNNB1, EOMES, JUN, FOS, HNF4a, HOXAs (e.g., HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA10, HOXA11, HOXA13), HOXBs (e.g., HOXB9), HOXCs (e.g., HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13), HOXDs (e.g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13), SNAIL-3, MYOD1, MYOG, NEUROD1-6 (e.g., NEUROD1, NEUROD2, NEUROD4, NEUROD6), PDX1, PU.1, SOX2, Nanog, Klf4, BCL-6, SOX9, STAT1-6, TBET, TCF, TEAD1-4 (e.g., TEAD1, TEAD2, TEAD3, TEAD4), TAF6L, CLOCK, CREB, GATA3, IRF7, MycC, NFkB, RORyt, RUNX1, SRF, TBX21, NFAT, MEF2D, and FoxP3.

[0126] In some cases, the intracellular domain is a transcription factor having a regulatory role in one or more immune cells (i.e., an immune cell regulatory transcription factor). Suitable immune cell regulatory transcription factors include, e.g., 2210012G02Rik, Akap81, App12, Arid4b, Arid5b, Ash11, Atf7, Atm, C430014K11Rik, Chd9, Dmtfl, Fos, Foxol, Foxpl, Hmboxl, Kdm5b, Klf2, Mga, M111, M113, Myst4, Pcgf6, Rev31, Scm14, Scp2, Smarca2, Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zebl, Zfml, Zfpl, Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652, Zfp827, Zhx2, Eomes, Arnt1, Bbx, Hbp1, Jun, Mef2d, Mterfd1, Nfat5, Nfe212, Nr1d2, Phf21a, Taf4b, Trf, Zbtb25, Zfp326, Zfp451, Zfp58, Zfp672, Egr2, Ikzf2, Tafld, Chracl, Dnajb6, Aplp2, Batf, Bhlhe40, Fosb, Hist1h1c, Hopx, Ifih1, Ikzf3, Lass4, Lin54, Mxd1, Mxi1, Prdm1, Prf1, Rora, Rpa2, Sap30, Stat2, Stat3, Taf9b, Tbx2l, Trpsl, Xbpl, Zeb2, Atf3, Cenpcl, Lass6, Rbl, Zbtb4l, Crem, Fos12, Gtf2b, Irf7, Maff, Nr4a1, Nr4a2, Nr4a3, Obfc2a, Rb12, Rel, Rybp, Sra1, Tgif1, Tnfaip3, Uhrf2, Zbtbl, Ccdcl24, Csda, E2f3, Epas1, Hif0, H2afz, Hif1a, Ikzf5, Irf4, Nsbp1, Pim1, Rfc2, Swap70, Tfblm, 2610036L11Rik, 5133400GO4Rik, Apitdl, Blm, Brcal, Bripl, Cld, C79407, Cenpa, Cfll, Clspn, Ddxl, Dsccl, E2f7, E2f8, Ercc61, Ezh2, Fenl, Foxml, Genl, Gsg2, H2afx, Hdacl, Hdgf, Hells, Histlhle, Hist3h2a, Hjurp, Hmgb2, Hmgb3, Irfl, Irf8, Kif22, Kif4, Ligl, Lmo2, Lnp, Mbd4, Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7, Myb12, Nei13, Nusapl, Orc61, Polal, Pola2, Pole, Pole2, Polh, Polr2f, Polr2j, Ppplr8, Prim2, Psmc3ip, Rad51, Rad51c, Rad541, Rfc3, Rfc4, Rnpsl, Rpal, Smarccl, Spic, Ssrpl, Taf9, Tfdpl, Tmpo, Topbp1, Trdmt1, Uhrf1, Wdhd1, Whsc1, Zbp1, Zbtb32, Zfp367, Carl, Polg2, Atr, Lefl, Myc, Nucb2, Satb1, Tafla, Ift57, Apexl, Chd7, Chtf8, Ctnnbl, Etv3, Irf9, Myb, Mybbpla, Pms2, Preb, Sp110, Stat1, Trp53, Zfp414, App, Cdk9, Ddbl, Hsf2, Lbr, Pa2g4, Rbmsl, Rfcl, Rfc5, Tada21, Tex261, Xrcc6, and the like.

[0127] In some cases, a transcription factor may be an artificial transcription factor (ATF) including but not limited to e.g., Zinc-finger-based artificial transcription factors (including e.g., those described in Sera T. Adv Drug Deliv Rev. 2009 61(7-8):513-26; Collins et al. Curr Opin Biotechnol. 2003 14(4):371-8; Onori et al. BMC Mol Biol. 2013 14:3 the disclosures of which are incorporated herein by reference in their entirety).

[0128] In some embodiments, the intracellular domain is a transcriptional activator. In some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following tetracycline-controlled transcriptional activator (tTA) amino acid sequence:

MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRH HTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFS LENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGL ELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO:33); and has a length of from about 245 amino acids to 252 amino acids (e.g., 248, 249, 250, 251, or 252 amino acids).

[0129] In some embodiments, the intracellular domain is a transcriptional activator. In some cases, the transcriptional activator is GAL4-VP16. In some cases, the transcriptional activator is GAL4-VP64. In some cases, the transcriptional activator is Tbx21. In some cases, the transcriptional activator is an engineered protein, such as a zinc finger or TALE based DNA binding domain fused to an effector domain such as VP64 (transcriptional activation) or KRAB (transcriptional repression). A variety of other transcriptional transactivators are known in the art is suitable for use.

[0130] In some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following GAL4-VP64 sequence:

MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERL EQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRIS ATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLGSDALDDFDLDMLGS (SEQ ID NO:34); and has a length of from 208 to 214 amino acids (e.g., 208, 209, 210, 211, 212, 213, or 214 amino acids).

Exemplary Embodiments

[0131] The following are non-limiting examples of suitable chimeric Notch receptor polypeptides.

[0132] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises a Notch receptor polypeptide that comprises, in order from N-terminus to C-terminus: i) Lin Notch Repeats A-C (an LNR segment); ii) a heterodimerization domain (an HD-N segment and an HD-C segment); iii) a TM domain; and comprises an 51 proteolytic cleavage site, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site. An example of such a Notch receptor polypeptide is depicted in FIG. 9A. In FIG. 9A, Lin Notch Repeats A-C (an LNR segment) have the following amino acid sequence:

PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDG HCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDC (SEQ ID NO:15); the heterodimerization domain (an HD-N segment and an HD-C segment) has the following amino acid sequence: AAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKR STVGWATSSLLPGTSGG ELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALA SLGSLNIPYKIE KSEPVEPPLP (SEQ ID NO:16), where the S1 proteolytic cleavage site includes the sequence RQRR (SEQ ID NO:37), and the S2 proteolytic cleavage site includes the sequence AV; and the TM domain has the following amino acid sequence: HLMYVAAAAFVLLFFVGCGVLLS (SEQ ID NO:17), where the S3 proteolytic cleavage site includes the sequence VLLS (SEQ ID NO:39).

[0133] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises a Notch receptor polypeptide that comprises, in order from N-terminus to C-terminus: i) an EGF repeat; ii) Lin Notch Repeats A-C (an LNR segment); iii) a heterodimerization domain (an HD-N segment and an HD-C segment); iv) a TM domain; and comprises an 51 proteolytic cleavage site, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site. An example of such a Notch receptor polypeptide is depicted in FIG. 9B. In FIG. 9B, the EGF repeat has the following amino acid sequence:

PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCH (SEQ ID NO:20); and the LNR segment, the heterodimerization domain, the TM domain, the S1 proteolytic cleavage site, the S2 proteolytic cleavage site, and the S3 proteolytic cleavage site are as depicted in FIG. 9A.

[0134] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises a Notch receptor polypeptide that comprises the following amino acid sequence:

TABLE-US-00010 (SEQ ID NO: 13) IPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR QLCIQKL;

where the TM domain is underlined; where the Notch receptor polypeptide comprises an S2 proteolytic cleavage site and an S3 proteolytic cleavage site; where the Notch receptor polypeptide has a length of 56 amino acids.

[0135] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an Fc receptor FcyIIIa (CD16A); b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) an Fc receptor FcyIIIa (CD16A); a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 9C. The locations of S1, S2, and S3 cleavage sites are depicted in FIG. 9A. In FIG. 9C, the CD16A has the following amino acid sequence:

MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFH NESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITIT QGLAVSTISSFFPPG (SEQ ID NO:42); the Notch receptor polypeptide has the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the following amino acid sequence:

TABLE-US-00011 (SEQ ID NO: 33) MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKR ALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAK VHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLE DQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICG LEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDM LPG.

[0136] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a cell surface antigen; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a CD19 polypeptide; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 10A. In FIG. 10A, the CD19 polypeptide has the following amino acid sequence:

RPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLF IFNVS QQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSS PSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRG PLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHL EITARPVLWHWLLRTGGWK (SEQ ID NO:44); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0137] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody specific for a cell surface antigen; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 10B. In FIG. 10B, the anti-CD19 scFv has the following amino acid sequence:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESG PGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN SKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:45); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0138] In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) an antibody specific for a cell surface antigen; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 10C. In FIG. 10C, the anti-CD19 scFv has the following amino acid sequence:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESG PGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN SKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:45); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9B; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0139] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody specific for a cell surface antigen, e.g., a cell surface antigen present on the surface of a cancer cell (e.g., a cancer-specific antigen); b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) an anti-mesothelin scFv; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 11. In FIG. 11, the anti-mesothelin scFv has the following amino acid sequence:

GSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQ KFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGS GGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLA SGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIKAS (SEQ ID NO:47); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9B; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0140] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody specific for a transcription factor; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an anti-myc scFv; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. Examples of such a chimeric Notch receptor polypeptide are depicted in FIG. 12A and 12B. In FIG. 12A and FIG. 12B, the anti-Myc scFv has the following amino acid sequence:

GSQVQLQQQVQLQESGGDLVKPGGSLKLSCAASGFTFSHYGMSWVRQTPDKRLEWVATIGSR GTYTHYPDSVKGRFTISRDNDKNALYLQMNSLKSEDTAMYYCARRSEFYYYGNTYYYSAMDY WGQGASVTVSSGGGGSGGGGSGGGGSDIVLTQSPAFLAVSLGQRATISCRASESVDNYGFSFM NWFQQKPGQPPKLLIYAISNRGSGVPARFSGSGSGTDFSLNIHPVEEDDPAMYFCQQTKEVPWT FGGGTKLEIK (SEQ ID NO:48); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0141] In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus: a) a LaG 9 nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 13A. In FIG. 13A, the LaG 9 nanobody has the following amino acid sequence:

MADVQLVESGGGLVQAGGSLRLSCAASGRTFSTSAMGWFRQAPGKEREFVARITWSAGYTAY SDSVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCASRSAGYSSSLTRREDYAYWGQGTQVT VS (SEQ ID NO:49); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0142] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a LaG 50 nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 13B. In FIG. 13B, the LaG 50 nanobody has the following amino acid sequence:

MADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVH ADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQV TVS (SEQ ID NO:50); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0143] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a LaG 18 nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 13C. In FIG. 13C, the LaG 18 nanobody has the following amino acid sequence:

MAQVQLVESGGGLVQTGGSLKLSCTASVRTLSYYHVGWFRQAPGKEREFVAGIHRSGESTFYA DSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAQRVRGFFGPLRSTPSWYDYWGQGTQV TVS (SEQ ID NO:51); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0144] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) a LaG 16/LaG 2 nanobody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a tTA transcription factor. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 13D. In FIG. 13D, the LaG 16/LaG 2 nanobody has the following amino acid sequence:

MAQVQLVESGGRLVQAGDSLRLSCAASGRTFSTSAMAWFRQAPGREREFVAAITWTVGNTILG DSVKGRFTISRDRAKNTVDLQMDNLEPEDTAVYYCSARSRGYVLSVLRSVDSYDYWGQGTQV TVSGGGGSGGGGSGGGGSMAQVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAP GKEREFVAAISWTGVSTYYADSVKGRFTISRDNDKNTVYVQMNSLIPEDTAIYYCAAVRARSFS DTYSRVNEYDYWGQGTQVTV (SEQ ID NO:52); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the tTA transcription factor has the amino acid sequence depicted in FIG. 9C.

[0145] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody specific for a cell surface antigen; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is Ga14-VP64 transcriptional activator. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 14. In FIG. 14, the anti-CD19 scFv has the following amino acid sequence:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGS GSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESG PGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN SKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO:45); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the Ga14-VP64 transcriptional activator has the following amino acid sequence:

TABLE-US-00012 (SEQ ID NO: 34) MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPL TRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFV QDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS AAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML GSDALDDFDLDMLGS.

[0146] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a DNA binding polypeptide. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an anti-CD19 scFv; b) a Notch receptor polypeptide comprising: i) an LNR segment; ii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iii) a TM domain where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a Zip(-) Gal4 DNA binding polypeptide. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 15. In FIG. 15, the anti-CD19 scFv has the amino acid sequence depicted in FIG. 14; the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9A; and the Zip(-) Gal4 DNA binding polypeptide has the following amino acid sequence:

TABLE-US-00013 (SEQ ID NO: 55) LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGG SGGSGGSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSP KTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKA LLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKG QRQLTVSAA.

[0147] In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an antibody; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is a transcriptional activator. In one non-limiting embodiment, a chimeric Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: a) an anti-mesothelin scFv; b) a Notch receptor polypeptide comprising: i) an EGF repeat; ii) an LNR segment; iii) a heterodimerization domain (an HD-N segment and an HD-C segment); and iv) a TM domain, where the Notch receptor polypeptide comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, where the intracellular domain is VP64 Zip(+) comprising an NLS. An example of such a chimeric Notch receptor polypeptide is depicted in FIG. 16. In FIG. 16, the anti-mesothelin scFv has the following amino acid sequence:

GSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQ KFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGS GGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYD (SEQ ID NO:56); the Notch receptor polypeptide includes the amino acid sequence depicted in FIG. 9B; and the VP64 Zip(+) transcriptional activator has the following amino acid sequence:

TABLE-US-00014 (SEQ ID NO: 57) PKKKRKVDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDAL DDFDLDMLGSGGSGGSGGSLEIEAAFLERENTALETRVAELRQRVQRLR NRVSQYRTRYGPLGGGK.

Force Sensor Cleavage Domain-Containing Chimeric Polypeptides

[0148] In some cases, the chimeric polypeptide is a force sensor cleavage domain-containing chimeric polypeptides (also referred to herein as "A2 chimeric polypeptides"). The term "force sensor cleavage domain", as used herein, refers to a polypeptide domain of a force sensitive protein that, upon the application of force, is cleavable, e.g., by a protease, including non-Notch force sensor cleavage domains. By "non-Notch force sensor cleavage domain", as used herein, is meant a cleavage domain of a force sensitive protein that is not, or is not derived from, a Notch protein. Such, non-Notch force sensor cleavage domains will not include a Notch negative regulatory region (NRR), Notch cleavage site(s) (e.g., S1, S2 or S3 sites) or any other portion of a Notch protein. However, in some instances, a non-Notch force sensor cleavage domain may be present in a polypeptide with other Notch-derived domains, such as domains of a Notch protein other than the Notch force sensitive cleavage domain Force sensor cleavage domains may be derived from force sensitive proteins from various species including but not limited to e.g., invertebrates (e.g., insects) and vertebrates (e.g., mammals such as mouse, rat, human and non-human primates), etc.

[0149] An A2 chimeric polypeptide comprises, from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising an antibody specific for a target antigen; b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site; c) a cleavable transmembrane domain; and d) an intracellular domain comprising a Notch intracellular signaling domain comprising a transcriptional activator. Binding of the antibody to the target antigen induces cleavage of the non-Notch force sensor cleavage domain at the proteolytic cleavage site, thereby releasing the intracellular domain The non-Notch force sensor cleavage domain is selected from the group consisting of: a von Willebrand Factor (vWF) cleavage domain, an amyloid-beta cleavage domain, a CD16 cleavage domain, a CD44 cleavage domain, a Delta cleavage domain, a cadherin cleavage domain, an ephrin-type receptor or ephrin ligand cleavage domain, a protocadherin cleavage domain, a filamin cleavage domain, a synthetic E cadherin cleavage domain, an interleukin-1 receptor type 2 (IL1R2) cleavage domain, a major prion protein (PrP) cleavage domain, a neuregulin cleavage domain and an adhesion-GPCR cleavage domain

[0150] In some cases, the proteolytic cleavage site is an ADAM family type protease cleavage site. In some cases, the ADAM family type protease cleavage site is an ADAM-13 type protease cleavage site. In some cases, the cleavable transmembrane domain comprises a .gamma.-secretase cleavage site. In some cases, the cleavable transmembrane domain is a Notch transmembrane domain In some cases, the Notch transmembrane domain comprises a .gamma.-secretase cleavage site. In some cases, the .gamma.-secretase cleavage site is a Notch S3 proteolytic cleavage site. In some cases, the Notch intracellular signaling domain is a drosophila Notch intracellular signaling domain In some cases, the non-Notch force sensor cleavage domain is a vWF cleavage domain In some cases, the vWF cleavage domain comprises a vWF A2 domain or a variant thereof. In some cases, the antibody is a nanobody, a diabody, a triabody, or a minibody, a F(ab').sub.2 fragment, a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody (sdAb).

[0151] An A2 chimeric polypeptide comprises a non-Notch force sensor cleavage domain, e.g., a vWF cleavage domain, an amyloid-beta cleavage domain, a CD16 cleavage domain, a CD44 cleavage domain, a Delta cleavage domain, a cadherin cleavage domain, an ephrin-type receptor or ephrin ligand cleavage domain, a protocadherin (e.g., drosophila fat) cleavage domain, a filamin cleavage domain, an E cadherin cleavage domain, an interleukin-1 receptor type 2 (i.e., IL1R2) cleavage domain, a major prion protein (i.e., PrP) cleavage domain, a neuregulin cleavage domain, an adhesion-GPCR cleavage domain, and homologs and variants thereof.

Cleavage Domains

[0152] An A2 chimeric polypeptide will generally include a force sensor cleavage domain that is not derived from a Notch polypeptide (i.e., a non-Notch force sensor cleavage domain) Such non-Notch force sensor cleavage domains will vary and may be derived from a force sensitive protein or homolog or variant thereof and will generally include at least one proteolytic cleavage site of the force sensitive protein.

[0153] Force sensor cleavage domains that may find use in an A2 chimeric polypeptides include vWF cleavage domains. Useful vWF cleavage domains will vary and may be derived from a vWF protein or homolog thereof and will generally include at least one proteolytic cleavage site of the vWF protein. Useful vWF proteolytic cleavage sites include ADAM family type protease cleavage sites, including e.g., ADAM-13 type protease cleavage sites. In some instances, a vWF polypeptide included in a chimeric polypeptide of the present disclosure may include a vWF A2 domain or a variant thereof. For example, in some instances, a vWF cleavage domain may include a mammalian vWF A2 domain, including but not limited to e.g., a human vWF A2 domain, a non-human primate vWF A2 domain, a rodent vWF A2 domain (e.g., a mouse vWF A2 domain, a rat vWF A2 domain, etc.) and the like. In some instances, a vWF cleavage domain may include a non-mammalian vWF A2 domain, including but not limited to e.g., an avian vWF A2 domain, a reptile vWF A2 domain, an amphibian vWF A2 domain, a fish vWF A2 domain, etc. Useful vWF A2 domains may include those vWF A2 domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring vWF A2 domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring vWF A2 domain (including e.g., mammalian and/or non-mammalian vWF A2 domains).

[0154] Useful human vWF cleavage domains may include e.g., amino acids 1480-1678 of the human vWF protein of UniProtKB ID P04275 or NCBI RefSeq ID NP_000543.2:

PGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYS YMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYM VTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSG EGLQI (SEQ ID NO:58), or a polypeptide having less than 100% sequence identity with the provided sequence, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with the provided sequence.

[0155] In some instances, a useful vWF cleavage domain may include the following amino acid sequence:

PGLLGVKKLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQY SYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVY MVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCS GEGLQI (SEQ ID NO:59), or a polypeptide having less than 100% sequence identity with the provided sequence, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with the provided sequence.

[0156] Useful mouse vWF cleavage domains may include e.g., amino acids 1480-1678 of the mouse vWF protein of UniProtKB ID Q8CIZ8:

PGIAGISSPGPKRKSMVLDVVFVLEGSDEVGEANFNKSKEFVEEVIQRMDVSPDATRISVLQYSY TVTMEYAFNGAQSKEEVLRHVREIRYQGGNRTNTGQALQYLSEHSFSPSQGDRVEAPNLVYMV TGNPASDEIKRLPGDIQVVPIGVGPHANMQELERISRPIAPIFIRDFETLPREAPDLVLQTCCSKEG LQLP (SEQ ID NO:60), or a polypeptide having less than 100% sequence identity with the provided sequence, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with the provided sequence.

[0157] Useful mouse vWF cleavage domains may include e.g., amino acids 183-381 of GenBank ID AAA82929.1:

PGIAGTLSPGPKRKSMVLDVVFVLEGSDEVGEANFNKSKEFVEEVIQRMDVSPDATRISVLQYS YTVTMEYAFNGAQSKEEVLRHVREIRYQGGNRTNTGQALQYLSEHSFSPSQGDRVEAPNLVYM VTGNPASDEIKRLPGDIQVVPIGVGPHANMQELERISRPIAPIFIRDFETLPREAPDLVLQTCCSKE GLQLP (SEQ ID NO:61), or a polypeptide having less than 100% sequence identity with the provided sequence, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with the provided sequence.

[0158] Useful vWF cleavage domains will vary in length, including e.g., where the overall length of the vWF cleavage domain is 1000 amino acids or less, including e.g., 900 amino acids or less, 800 amino acids or less, 700 amino acids or less, 600 amino acids or less, 500 amino acids or less, 400 amino acids or less, 300 amino acids or less or 200 amino acids or less. In some instances, the subject vWF cleavage domain may range from less than 150 to more than 1000 amino acid in length, including but not limited to e.g., from 150 to 1000, from 150 to 900, from 150 to 800, from 150 to 700, from 150 to 600, from 150 to 500, from 150 to 400, from 150 to 350, from 150 to 300, from 150 to 275, from 150 to 250, from 150 to 225, from 150 to 200, or the like.

[0159] In some instances, a vWF cleavage domain may include sequence of a vWF protein in the N- and/or C-terminal direction adjacent to a vWF A2 domain, including up to 100 amino acids or more in the N- and/or C-terminal direction adjacent to the A2 domain, including but not limited to e.g., 100 amino acids or less, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, 10 amino acids or less, etc., in the N- and/or C-terminal direction adjacent to a vWF A2 domain

[0160] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include amyloid-beta cleavage domains. Useful amyloid-beta cleavage domains will vary and may be derived from an amyloid-beta protein (e.g., amyloid-beta A4 protein, amyloid precursor protein (APP), etc.) or homolog thereof and will generally include at least one proteolytic cleavage site of the amyloid-beta protein. In some instances, an amyloid-beta polypeptide included in a chimeric polypeptide of the present disclosure may be a mammalian amyloid-beta cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like amyloid-beta cleavage domains and homologs and variants thereof. Useful amyloid-beta cleavage domains may include those amyloid-beta cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring amyloid-beta cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring amyloid-beta cleavage domain (including e.g., mammalian and/or non-mammalian amyloid-beta cleavage domains.

[0161] Useful amyloid-beta cleavage domains may include e.g., those derived from accession number RefSeq NP_001129603.1 or a homolog or variant thereof, including e.g.,:

TABLE-US-00015 (SEQ ID NO: 62) MVSKGEEDNSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDD EDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVRVPTTAA STPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAER QAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLND RRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVR MVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDEL LQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFS LDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKT EEISEVNLDAEFRHDSGYEVHHQKLVFFAEDVGSNKGR,

or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0162] Subject amyloid-beta cleavage domains may be derived from or include a portion of a sequence from a wide variety of amyloid-beta protein sequences.

[0163] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include CD16 cleavage domains. Useful CD16 cleavage domains will vary and may be derived from a CD16 protein (e.g., low affinity immunoglobulin gamma Fc region receptor III protein) or homolog thereof and will generally include at least one proteolytic cleavage site of the CD16 protein. In some instances, a CD16 polypeptide included in a chimeric polypeptide of the present disclosure may be a mammalian CD16 cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like CD16 cleavage domains and homologs and variants thereof. Useful CD16 cleavage domains may include those CD16 cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring CD16 cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring CD16 cleavage domain (including e.g., mammalian CD16 cleavage domains).

[0164] Useful CD16 cleavage domains may include e.g., those derived from accession number RefSeq NP_001121065.1 or a homolog or variant thereof, including e.g.,:

GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAAT VDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQ NGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQV R (SEQ ID NO:63), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0165] Subject CD16 cleavage domains may be derived from or include a portion of a sequence from a wide variety of CD16 protein sequences.

[0166] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include CD44 cleavage domains. Useful CD44 cleavage domains will vary and may be derived from a CD44 protein (e.g., CD44 antigen isoform a precursor) or homolog thereof and will generally include at least one proteolytic cleavage site of the CD44 protein. In some instances, a CD44 polypeptide included in a chimeric polypeptide of the present disclosure may be a mammalian CD44 cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like CD44 cleavage domains and homologs and variants thereof. Useful CD44 cleavage domains may include those CD44 cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring CD44 cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring CD44 cleavage domain (including e.g., mammalian and/or non-mammalian CD44 cleavage domains).

[0167] Useful CD44 cleavage domains may include e.g., those derived from accession number RefSeq NP_033981.2 or a homolog or variant thereof, including e.g.,:

PRHSKSHAAAQKQNNWIWSWFGNSQSTTQTQEPTTSATTALMTTPETPPKRQEAQNWFSWLFQ PSESKSHLHTTTKMPGTESNTNPTGWEPNEENEDETDKYPSFSGSGIDDDEDFISSTIASTPRVSA RTEDNQDWTQWKPNHSNPEVLLQTTTRMADIDRISTSAHGENWTPEPQPPFNNHEYQDEEETP HATSTTPNSTAEAAATQQETWFQNGWQGKNPPTPSEDSHVTEGTTASAHNNHPSQRITTQSQED VSWTDFFDPISHPMGQGHQTESKDTDSSHSTTLQPTAAPNTHLVEDLNRTGPLSVTTPQSHSQNF STLHGEPEEDENHPTTSILPSSTKSGAKDARRGGSLPTDTTTSVEGYTFQYPDTMENGTLFPVTP AKTEVFGETEVTLATDSNVNVDGSLPGDRDSSKDSRGSSRTVTHGSELAGHSSANQDSGVTTTS GPMRRPQIPER (SEQ ID NO:64), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0168] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include Delta cleavage domains. Useful Delta cleavage domains will vary and may be derived from a Delta protein (e.g., Drosophila neurogenic locus protein delta) or homolog thereof and will generally include at least one proteolytic cleavage site of the Delta protein. In some instances, a Delta polypeptide included in a chimeric polypeptide of the present disclosure may be a mammalian Delta cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like Delta cleavage domains and homologs and variants thereof. Useful Delta cleavage domains may include those Delta cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring Delta cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring Delta cleavage domain (including e.g., mammalian and/or non-mammalian Delta cleavage domains).

[0169] Useful Delta cleavage domains may include e.g., those derived from accession number GenBank CAA29617.1 or a homolog or variant thereof, including e.g.,:

PRDEESYDSVTFDAHQYGATTQARADGLANAQVR (SEQ ID NO:65), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0170] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include cadherin cleavage domains. Useful cadherin cleavage domains will vary and may be derived from a cadherin protein (e.g., cadherin-1 preproprotein) or homolog thereof and will generally include at least one proteolytic cleavage site of the cadherin protein. In some instances, a cadherin polypeptide included in an A2 chimeric polypeptide may be a mammalian cadherin cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like cadherin cleavage domains and homologs and variants thereof. Useful cadherin cleavage domains may include those cadherin cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring cadherin cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring cadherin cleavage domain (including e.g., mammalian and/or non-mammalian cadherin cleavage domains).

[0171] Useful cadherin cleavage domains may include e.g., those derived from accession number RefSeq NP_033994.1 or a homolog or variant thereof, including e.g.,:

AEMDREDAEHVKNSTYVALIIATDDGSPIATGTGTLLLVLLDVNDNAPIPEPRNMQFCQRNPQP HIITILDPDLPPNTSPFTAELTHGASVNWTIEYNDAAQESLILQPRKDLEIGEYKIHLKLADNQNK DQVTTLDVHVCDCEGTVNNCMKAGIVAAGLQVR (SEQ ID NO:66), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0172] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include ephrin-type receptor and ephrin ligand cleavage domains. Useful ephrin-type receptor and ephrin ligand cleavage domains will vary and may be derived from an ephrin-type receptor and ephrin ligand proteins (e.g., ephrin type-B receptor 2, ephrin-B2 precursor, ephrin-A2 precursor, etc.) or homolog thereof and will generally include at least one proteolytic cleavage site of the protein. In some instances, an ephrin-type receptor or ephrin ligand polypeptide included in an A2 chimeric polypeptide may be a mammalian ephrin-type receptor or ephrin ligand cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like ephrin-type receptor or ephrin ligand cleavage domains and homologs and variants thereof. Useful ephrin-type receptor and ephrin ligand cleavage domains may include those ephrin-type receptor and ephrin ligand cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring ephrin-type receptor or ephrin ligand cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring ephrin-type receptor or ephrin ligand cleavage domain (including e.g., mammalian and/or non-mammalian ephrin-type receptor and ephrin ligand cleavage domains).

[0173] Useful ephrin-type receptor and ephrin ligand cleavage domains may include e.g., those derived from accession numbers RefSeq NP_001277682.1, RefSeq NP_034241.2, RefSeq NP_031935.3, or a homolog or variant thereof, including e.g.,:

NGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLYCNGDGEWLVPIGRCMCKAGFEAVENG TVCRGCPSGTFKANQGDEACTHCPINSRTTSEGATNCVCRNGYYRADLDPLDMPCTTIPSAPQA VISSVNETSLMLEWTPPRDSGGREDLVYNIICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYI SDLLAHTQYTFEIQAVNGVTDQSPFSPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPD QPNGVILDYELQYYEKQELSEYNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGK MYFQTMTEAEYQTSIKEKLPR (SEQ ID NO:67), APSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKQELSEYNATAIKSPTNTVTVQGL KAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIKEKLPR (SEQ ID NO:68), MAMARSRRDSVWKYCWGLLMVLCRTAISRSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDII CPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCARPDQDVKFTIKFQEFSPNLWGL EFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVGQDAS SAGSARNHGPTRRPELEA GTNGRSSTTSPFVKPNPGSSTDGNSAGHSGNNLLGSEVALFAR (SEQ ID NO:69), VYVRPTNETLYEAPEPIFTSNSSCSGLGGCHLFLTTVPVLWSLLGSR (SEQ ID NO:70), or GQDASSAGSARNHGPTRRPELEAGTNGRSSTTSPFVKPNPGSSTDGNSAGHSGNNLLGSEVALF AR (SEQ ID NO:71) or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0174] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include protocadherin cleavage domains. Useful protocadherin cleavage domains will vary and may be derived from a protocadherin protein (e.g., Drosophila fat) or homolog thereof and will generally include at least one proteolytic cleavage site of the protocadherin protein. In some instances, a protocadherin polypeptide included in an A2 chimeric polypeptide may be a mammalian protocadherin cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like protocadherin cleavage domains and homologs and variants thereof. Useful protocadherin cleavage domains may include those protocadherin cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring protocadherin cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring protocadherin cleavage domain (including e.g., mammalian and/or non-mammalian protocadherin cleavage domains).

[0175] Useful protocadherin cleavage domains may include e.g., those derived from accession number RefSeq NP_477497.1 or a homolog or variant thereof, including e.g.,:

DNQQMRERRAVSNFSTASQIYEAPKMLSMLFRTYKDQGQILYAATNQMFTSLSLREGRLVYYS KQHLTINMTVQETSTLNDGKWHNVSLFSESRSLRLIVDGRQVGDELDIAGVHDFLDPYLTILNV GGEAFVGCLANVTVNNELQPLNGSGSIFPEVRYHGKIESGCRGDIGQDAAQVADPLSIGFTLVIV FFVILVVAILGSYVIYRFR (SEQ ID NO:72), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0176] Force sensor cleavage domains that may find use in the instant chimeric polypeptides include filamin cleavage domains. Useful filamin cleavage domains will vary and may be derived from a filamin protein (e.g., filamin-A isoform 2) or homolog thereof and will generally include at least one proteolytic cleavage site of the filamin protein. In some instances, a filamin polypeptide included in a chimeric polypeptide of the present disclosure may be a mammalian filamin cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like filamin cleavage domains and homologs and variants thereof. Useful filamin cleavage domains may include those filamin cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring filamin cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring filamin cleavage domain (including e.g., mammalian and/or non-mammalian filamin cleavage domains).

[0177] Useful filamin cleavage domains may include e.g., those derived from accession number RefSeq NP_001104026.1 or a homolog or variant thereof, including e.g.,:

MACKMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQ KESTLHLVLRLRGGELGGSGGSGEGRVKESITRRRRAPSVANVGSHSDLSLKIPEISIQDMTAQV TSPSGKTHEAEIVEGENHTYSIRFVPAEMGTHTVSVKYKGQHVPGSPFQFTVGPLGEGGAHKVR AGGPGLERAEAGVPAEFSIWTREAGAGGLAIAVEGPSKAEISFEDRKDGSSGVAYVVQEPGDYE VSVKFNEEHIPDSPFVVPVASPSSGGSGGTMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIP PDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGKCLER (SEQ ID NO:73) or MACKMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQ KESTLHLVLRLRGGELGGSGGPTFRSSLFLWVRPGGSGGSGPLGEGGAHKVRAGGPGLERAEA GVPAEFSIWTREAGAGGLAIAVEGPSKAEISFEDRKDGSCGVAYVVQEPGDYEVSVKFNEEHIP DSPFVVPVASPSSGGSGGTMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAG KQLEDGRTLSDYNIQKESTLHLVLRLRGGKCLER (SEQ ID NO:74), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0178] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include E cadherin cleavage domains. Useful E cadherin cleavage domains will vary and may be derived from an E cadherin protein or homolog thereof or recombinant variants thereof (e.g., EcadTS) and will generally include at least one proteolytic cleavage site of the E cadherin protein. In some instances, an E cadherin polypeptide included in an A2 chimeric polypeptide may be a mammalian E cadherin cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like E cadherin cleavage domains and homologs and variants thereof. Useful E cadherin cleavage domains may include those E cadherin cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring E cadherin cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring E cadherin cleavage domain (including e.g., mammalian and/or non-mammalian E cadherin cleavage domains).

[0179] Useful E cadherin cleavage domains may include e.g., those derived from accession number GenBank AID22384.1 or a homolog or variant thereof, including e.g.,:

MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDGTNTINLEVKEGAPLPFSY DILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTFEDKGIVKVKSDISMEEDSFIYEIHL KGENFPPNGPVMQKKTTGWDASTERMYVRDGVLKGDVKHKLLLEGGGHHRVDFKTIYRAKK AVKLPDYHFVDHRIEILNHDKDYNKVTVYESAVARNSTDGMDELYKGPGGAGPGGAGPGGAG PGGAGPGGAGPGGAGPGGAGPGGAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDA TYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANF KIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGI TLGMDELYK (SEQ ID NO:75), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0180] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include interleukin-1 receptor type 2 (i.e. IL1R2) cleavage domains. Useful IL1R2 cleavage domains will vary and may be derived from an IL1R2 protein (e.g., interleukin-1 receptor type 2 isoform 1 precursor) or homolog thereof and will generally include at least one proteolytic cleavage site of the IL1R2 protein. In some instances, an IL1R2 polypeptide included in A2 chimeric polypeptide may be a mammalian IL1R2 cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like IL1R2 cleavage domains and homologs and variants thereof. Useful IL1R2 cleavage domains may include those IL1R2 cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring IL1R2 cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring IL1R2 cleavage domain (including e.g., mammalian and/or non-mammalian IL1R2 cleavage domains).

[0181] Useful IL1R2 cleavage domains may include e.g., those derived from accession number RefSeq NP_004624.1 or a homolog or variant thereof, including e.g.,:

AARSCRFRGRHYKREFRLEGEPVALRCPQVPYWLWASVSPRINLTWHKNDSARTVPGEEETRM WAQDGALWLLPALQEDSGTYVCTTRNASYCDKMSIELRVFENTDAFLPFISYPQILTLSTSGVLV CPDLSEFTRDKTDVKIQWYKDSLLLDKDNEKFLSVRGTTHLLVHDVALEDAGYYRCVLTFAHE GQQYNITRSIELRIKKKKEETIPVIISPLKTISASLGSRLTIPCKVFLGTGTPLTTMLWWTANDTHIE SAYPGGRVTEGPRQEYSENNENYIEVPLIFDPVTREDLHMDFKCVVHNTLSFQTLRTTVKEASST FSGR (SEQ ID NO:76), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0182] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include major prion protein (i.e. PrP) cleavage domains. Useful PrP cleavage domains will vary and may be derived from a PrP protein (e.g., major prion protein precursor) or homolog thereof and will generally include at least one proteolytic cleavage site of the PrP protein. In some instances, a PrP polypeptide included in an A2 chimeric polypeptide may be a mammalian PrP cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like PrP cleavage domains and homologs and variants thereof. Useful PrP cleavage domains may include those PrP cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring PrP cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring PrP cleavage domain (including e.g., mammalian and/or non-mammalian PrP cleavage domains).

[0183] Useful PrP cleavage domains may include e.g., those derived from accession number RefSeq NP_035300.1 or a homolog or variant thereof, including e.g.,:

KRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGTWGQPHGGGWGQPHGGSWGQPHGGSWGQP HGGGWGQGGGTHNQWNKPSKPKTNLKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGND WEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKM MERVVEQMCVTQYQKESQAYYDGRRSSSTVLFSSPPVILLISFLIFLIVGR (SEQ ID NO:77), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0184] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include neuregulin cleavage domains. Useful neuregulin cleavage domains will vary and may be derived from a neuregulin protein (e.g., pro-neuregulin-1, membrane-bound isoform isoform 111-3, neuregulin Nrg1 (type III), etc.) or homolog thereof and will generally include at least one proteolytic cleavage site of the neuregulin protein. In some instances, a neuregulin polypeptide included in an A2 chimeric polypeptide may be a mammalian neuregulin cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like neuregulin cleavage domains and homologs and variants thereof. Useful neuregulin cleavage domains may include those neuregulin cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring neuregulin cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring neuregulin cleavage domain (including e.g., mammalian and/or non-mammalian neuregulin cleavage domains).

[0185] Useful neuregulin cleavage domains may include e.g., those derived from accession number RefSeq NP_001309136.1 or a homolog or variant thereof, including e.g.,:

GDRCQNYVMASFYKHLGIEFMEAEELYQKRVLTITGICIAR (SEQ ID NO:78), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0186] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include adhesion-GPCR cleavage domains. Useful adhesion-GPCR cleavage domains will vary and may be derived from an adhesion-GPCR protein (e.g., Drosophila Flamingo) or homolog thereof and will generally include at least one proteolytic cleavage site of the adhesion-GPCR protein. In some instances, an adhesion-GPCR polypeptide included in an A2 chimeric polypeptide may be a mammalian adhesion-GPCR cleavage domain or a variant thereof, including but not limited to e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), and the like adhesion-GPCR cleavage domains and homologs and variants thereof. Useful adhesion-GPCR cleavage domains may include those adhesion-GPCR cleavage domains that are naturally occurring or non-natural variants thereof, including e.g., domains having less than 100% sequence identity with a naturally occurring adhesion-GPCR cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring adhesion-GPCR cleavage domain (including e.g., mammalian and/or non-mammalian adhesion-GPCR cleavage domains).

[0187] Useful adhesion-GPCR cleavage domains may include e.g., those derived from accession number GenBank BAA84069.1 or a homolog or variant thereof, including e.g.,:

PRNPQCVRWNSFTNRWTRLGCQTEIPDFDGDFNPAAQQAILVNCSCTHISSYAVIVDVIDPEDIPE PSLLVQR (SEQ ID NO:79) or

[0188] ITYPSEQMQQSEQVVYRSLGSPHLAQPIKLQMWLDVDSARFGPRSNPQCVRWNSFTNRWTRLG CQTEIPDFDGDFNPAAQQAILVNCSCTHISSYAVIVDVIDPEDIPEPSLLVQR (SEQ ID NO:80), or a polypeptide having less than 100% sequence identity with the preceding sequence or another sequence derived from the protein of the provided accession number, including e.g., at least 99% sequence identity, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% or at least 60% sequence identity with one or more of the provided sequences.

[0189] Force sensor cleavage domains that may find use in an A2 chimeric polypeptide include synthetic cleavage domains, including e.g., flagellin-derived cleavage domains. Useful flagellin-derived cleavage domains will vary and may be derived from a flagellin protein or homolog thereof and will generally include at least one proteolytic cleavage site. Useful synthetic cleavage domains may include, e.g., PRGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGARR (SEQ ID NO:81) including e.g., domains having less than 100% sequence identity with synthetic cleavage domain, including one or more of the domains provided herein, such as less than 100% but at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with a naturally occurring synthetic cleavage domain (including e.g., the synthetic domain provided above).

[0190] The cleavage domains (including e.g., those provided above) may be included in an A2 chimeric polypeptide at any convenient and appropriate location that may vary, e.g., depending on the length of the cleavage domain, the inclusion or exclusion of additional domains (i.e., domains besides the cleavage domain) of the protein from which the cleavage domain is derived in the chimeric polypeptide, the presence or absence of other domains, e.g., as described herein, within the A2 chimeric polypeptide, and the like. In some embodiments, the cleavage domain may be positioned within the A2 chimeric polypeptide essentially as described for the vWF cleavage domain(s) described herein. In some embodiments, a cleavage domain may be inserted within the A2 chimeric polypeptide following a Notch domain, e.g., following and/or adjacent to a PPANVKYV (SEQ ID NO:82) of a Notch domain), or the like. Useful force sensor cleavage domains will vary in length, including e.g., where the overall length of the force sensor cleavage domain is 1000 amino acids or less, including e.g., 900 amino acids or less, 800 amino acids or less, 700 amino acids or less, 600 amino acids or less, 500 amino acids or less, 400 amino acids or less, 300 amino acids or less, 200 amino acids or less, 100 amino acids or less or 50 amino acids or less. In some instances, the subject force sensor cleavage domain may range from less than 40 to more than 1000 amino acid in length, including but not limited to e.g., from 40 to 1000, from 50 to 1000, from 75 to 1000, from 100 to 1000, from 125 to 1000, from 150 to 1000, from 150 to 900, from 150 to 800, from 150 to 700, from 150 to 600, from 150 to 500, from 150 to 400, from 150 to 350, from 150 to 300, from 150 to 275, from 150 to 250, from 150 to 225, from 150 to 200, from 40 to 900, from 40 to 800, from 40 to 700, from 40 to 600, from 40 to 500, from 40 to 400, from 40 to 350, from 40 to 300, from 40 to 275, from 40 to 250, from 40 to 225, from 40 to 200, from 40 to 100 or the like.

[0191] In some instances, a force sensor cleavage domain may include sequence of a force sensitive protein in the N- and/or C-terminal direction adjacent to a force sensor cleavage domain, including up to 100 amino acids or more in the N- and/or C-terminal direction adjacent to the force sensor cleavage domain, including but not limited to e.g., 100 amino acids or less, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, 10 amino acids or less, etc., in the N- and/or C-terminal direction adjacent to a force sensor cleavage domain

Transmembrane Domains

[0192] An A2 chimeric polypeptide will generally include a transmembrane domain Useful transmembrane domains include those having a proteolytic cleavage site (i.e., cleavable transmembrane domains). Proteolytic cleavage of a cleavable transmembrane domain of an A2 chimeric polypeptide will generally be prevented prior to cleavage of the chimeric polypeptide at the force sensor cleavage domain. Put another way, within an A2 chimeric polypeptide, cleavage at a cleavable transmembrane domain cleavage site may be blocked, e.g., blocked by one or more ectodomains of the chimeric polypeptide, until the chimeric polypeptide is cleaved at a proteolytic cleavage site within the force sensor cleavage domain Thus, cleavage of an A2 chimeric polypeptide at a proteolytic cleavage site within the force sensor cleavage domain may thereby expose a cleavage site of the cleavable transmembrane domain, i.e., exposing an otherwise inaccessible transmembrane domain cleavage site to cleavage by a protease. The process whereby removal of one or more ectodomains is required for cleavage of a cleavable transmembrane domain may also be referred to as ectodomain shedding. As such, in some instances, ectodomain shedding by cleavage at a force sensor cleavage domain may provide for subsequent cleavage at a transmembrane domain cleavage site.

[0193] Various cleavable transmembrane domains may find use in an A2 chimeric polypeptide. For example, in some instances, useful cleavable transmembrane domains include those having, either naturally or artificially, a y-secretase cleavage site. Substrates of y-secretase include e.g., Alcadein .alpha., Alcadein .gamma. (calsyntenin), APLP1, APLP2, ApoER2, APP, A.beta.PP, Betacellulin (BTC), Betaglycan, CD43, CD44, CSF1R, CX3CL1 (fractalkine), CXCL16, DCC, Deltal, Desmoglein-2, DNER, Dystroglycan, E-cadherin, EpCAM, EphA4, EphB2 EphrinB1, EphrinB2, ErbB4, GHR, HLA, HLA-A2, IFNaR2, IGF-1R, IL-1R1, IL-1R2, IL6R, IR, Ire1.beta., Ire1.alpha., Jagged2, KCNE1, KCNE2, KCNE3, KCNE4, Klotho, L1, LAR, LRP1 (LDLR), LRP1B, LRP2 (megalin), LRP6, MUC1, Nav-.beta.1, Nav-.beta.2, Nav-.beta.3, Nav-.beta.4, N-cadherin, Nectin-1.alpha., Neuregulin-1, Neuregulin-2, Notch1, Notch2, Notch3, Notch4, NPR-C, NRADD, p75-NTR, PAM, PLXDC2, Polyductin (PKHD1), Protocadherin-.alpha.4 (Pcdh-.alpha.4), Protocadherin-.gamma.-C3 (Pcdh-.gamma.C3), PTP-LAR, Ptprz, RAGE, ROBO1, RPTP.kappa., RPTP.mu., SorC3, SorCS1b, SorLA (LR11), Sortilin, Syndecan-1, Syndecan-2, Syndecan-3, Tie1, Tyrosinase, TYRP1, TYRP2, Vasorin, VE-cadherin, VEGF-R1, VGSC beta2, VLDLR, as well as those described in Bed & Sanders (Cell Mol Life Sci. (2008) 65(9):1311-1334) and Haapasalo & Kovacs (J Alzheimers Dis. (2011) 25(1):3-28); the disclosures of which are incorporated herein by reference in their entirety.

[0194] Useful transmembrane domains include but are not limited to Notch transmembrane domains, including e.g., invertebrate and vertebrate Notch transmembrane domains, including e.g., insect (e.g., drosophila) Notch transmembrane domains, mammalian (e.g., human, non-human primate, rodent (e.g., mouse, rat, etc.), etc.) Notch transmembrane domains, and the like. Notch transmembrane domains are generally cleavable transmembrane domains, as described herein, and may, e.g., include a y-secretase cleavage site, including natural and modified y-secretase cleavage sites, including e.g., a Notch S3 proteolytic cleavage site.

[0195] Useful Notch transmembrane domains include but are not limited to e.g., Notch 1, Notch 2, Notch 3 and Notch 4 transmembrane domains. Non-limiting examples of Notch transmembrane domains include but are not limited to e.g., FMYVAAAAFVLLFFVGCGVLL (SEQ ID NO: 83), LLYLLAVAVVIILFIILLGVI (SEQ ID NO:84), LPLLVAGAVLLLVILVLGVMV (SEQ ID NO:85), PVLCSPVAGVILLALGALLVL (SEQ ID NO:86), LMYVAAAAFVLLFFVGCGVLL (SEQ ID NO:87), LLYLLAVAVVIILFFILLGVI (SEQ ID NO:88), LLPLLVAGAVFLLIIFILGVM (SEQ ID NO:89), PILCSPVVGVLLLALGALLVL (SEQ ID NO:90), LHLMYVAAAAFVLLFFVGCGVLL (SEQ ID NO:91), LLYLLAVAVVIILFLILLGVI (SEQ ID NO:92), LPLLVAGAVFLLVIFVLGVMV (SEQ ID NO:93), and variants thereof.

[0196] A Notch transmembrane domain or a portion thereof utilized in an A2 chimeric polypeptide may include an S3 cleavage site (i.e., a gamma-secretase cleavage site). As such, an S3 proteolytic cleavage site can be located within the TM domain The S3 proteolytic cleavage site may be cleaved by gamma-secretase (y-secretase). A .gamma.-secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between the Gly and the Val. For example, in some cases, an S3 proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO:31), where cleavage occurs between the "GV" sequence. In some cases, an S3 proteolytic cleavage site comprises the amino acid sequence GCGVLLS (SEQ ID NO:32).

Extracellular Antigen-Binding Domains

[0197] An A2 chimeric polypeptide comprises an antibody specific for a target antigen. The antibody can be any antigen-binding antibody-based polypeptide, a wide variety of which are known in the art. In some instances, the antibody is a monoclonal antibody, a single chain Fv (scFv), a Fab, etc. Other suitable antibodies include, e.g., cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and "camelized" antibody variable domains.

[0198] In some cases, the antigen-binding domain (antibody) is specific for a cancer antigen, i.e., an antigen expressed by (synthesized by) a neoplasia or cancer cell, i.e., a cancer cell associated antigen or a cancer (or tumor) specific antigen.

[0199] A cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancer cell associated antigen may also be expressed by a non-cancerous cell.

[0200] A cancer cell specific antigen can be an antigen specific for cancer and/or a particular type of cancer or cancer cell including e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancer (or tumor) specific antigen is generally not expressed by non-cancerous cells (or non-tumor cells). In some instances, a cancer (or tumor) specific antigen may be minimally expressed by one or more non-cancerous cell types (or non-tumor cell types). By "minimally expressed" is meant that the level of expression, in terms of either the per-cell expression level or the number of cells expressing, minimally, insignificantly or undetectably results in binding of the specific binding member to non-cancerous cells expressing the antigen.

[0201] An A2 chimeric polypeptide may, in some cases, target a surface expressed antigen. As used herein the term "surface expressed antigen" generally refers to antigenic proteins that are expressed at least partially extracellularly such that at least a portion of the protein is exposed outside the cells and available for binding with a binding partner. Essentially any surface expressed protein may find use as a target of an A2 chimeric polypeptide. Non-limiting examples of surface expressed antigens include but are not limited to e.g., CD19, CD20, CD30, CD38, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), IL-13R-a2, GD2, and the like. Surface expressed antigens that may be targeted also include but are not limited to e.g., those specifically targeted in conventional cancer therapies, including e.g., those targets of the targeted cancer therapeutics described herein.

[0202] In some instances, the antibody of an A2 chimeric polypeptide may target a cancer-associated antigen. In some instances, the antibody is specific for a cancer associated antigen. Non-limiting examples of cancer associated antigens include but are not limited to e.g., CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Cancer-associated antigens also include, e.g., 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin .alpha.5B1, integrin .alpha.v.beta.3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R .alpha., PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-.beta., TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0203] The antibody portion of an A2 chimeric polypeptide can specifically bind an antigen that is associated with an inflammatory disease. Non-limiting examples of antigens associated with inflammatory disease include, e.g., AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (.alpha. chain of IL-2 receptor), CD3, CD4, CDS, IFN-.alpha., IFN-.gamma., IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin .alpha.4, integrin .alpha.4.beta.7, LFA-1 (CD11a), myostatin, OX-40, scleroscin, SOST, TGF beta 1, TNF-.alpha., and VEGF-A.

[0204] The antibody portion of an A2 chimeric polypeptide can specifically bind an autoantigen.

Generating a Synthetic Immune Cell

[0205] A synthetic immune cell of the present disclosure can be generated using well-established methods. As noted above, a synthetic immune cell of the present disclosure is a genetically modified immune cell that is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator.

[0206] The one or more nucleic acids can be expression vectors. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, the vector is a lentivirus vector. Also suitable are transposon-mediated vectors, such as piggyback and sleeping beauty vectors.

[0207] In some embodiments, the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus (AAV) construct, a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a recombinant lentiviral construct, etc. In some cases, a nucleic acid comprising a nucleotide sequence encoding a chimeric receptor polypeptide is a recombinant lentivirus vector. In some cases, a nucleic acid comprising a nucleotide sequence encoding a chimeric receptor polypeptide is a recombinant AAV vector. In some cases, a nucleic acid comprising a nucleotide sequence encoding a cytokine is a recombinant lentivirus vector. In some cases, a nucleic acid comprising a nucleotide sequence encoding a cytokine is a recombinant AAV vector.

[0208] The present disclosure provides a composition comprising: a) a first recombinant expression vector comprising a nucleotide encoding a chimeric polypeptide, where the chimeric polypeptide comprises: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a second recombinant expression vector comprising a nucleotide encoding a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator. In some cases, the chimeric polypeptide is a chimeric Notch polypeptide, as described above. In some cases, the cytokine is a variant IL-2 polypeptide, as described above.

[0209] An immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding the chimeric polypeptide and the cytokine, where the genetic modification is accomplished by introducing into the immune cell the one or more nucleic acids. Methods of introducing a nucleic acid into a host cell (e.g., an immune cell) are known in the art and include, e.g., electroporation, transduction, transfection, etc.

Compositions

[0210] The present disclosure provides a composition comprising: a) a synthetic immune cell as described above; and b) a second immune cell, where the second immune cell is a cytotoxic T cell (CTL). As described above, a synthetic immune cell is a genetically modified, in vitro immune cell, wherein the immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, where the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator.

[0211] In some cases, the second immune cell is a CTL that is genetically modified to express on its surface an exogenous T-cell receptor (TCR), wherein the exogenous TCR is specific for a target antigen. In some cases, the second immune cell is a CTL that is genetically modified to express on its surface a chimeric antigen receptor (CAR), wherein the CAR is specific for a target antigen. In some cases, the second immune cell is a CTL that is genetically modified to express a bispecific T-cell engager (BiTE), wherein the BiTE comprises: i) a first antigen-binding region specific for CD3; and ii) a second antigen-binding region specific for a target antigen other than CD3. In some cases, the target antigen to which the TCR, the CAR, or the BiTE binds is a different antigen than the antigen to which the antibody present in the chimeric polypeptide of the synthetic immune cell binds. In some cases, the target antigen to which the TCR, the CAR, or the BiTE binds is the same antigen as the antigen to which the antibody present in the chimeric polypeptide of the synthetic immune cell binds. In some cases, the TCR, the CAR, or the BiTE is specific for a cancer-associated antigen, and the antibody present in the chimeric polypeptide is specific for a cancer-associated antigen.

[0212] In some cases, the second immune cell is a CTL that is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding a CAR. The terms "chimeric antigen receptor" and "CAR", used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014/127261 and US Patent Application No. 2015/0368342, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19-4-1BB-CD3.zeta. CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0213] In some cases, the CAR comprises: a) an extracellular domain comprising an antigen-binding domain (e.g., an antibody, such as a scFv or a nanobody); b) a transmembrane region; and c) an intracellular signaling domain In some cases, the intracellular signaling domain comprises: i) a signaling domain from the zeta chain of human CD3; and ii) one or more costimulatory polypeptides. In some cases, the one or more costimulatory polypeptides is selected from CD28, 4-1BB, and OX-40. The CAR is in some cases a single polypeptide chain CAR. In some cases, the CAR comprises 2 polypeptide chains; for example, in some cases, the 2 polypeptide chains comprise dimerization domains that dimerize in the presence of a small molecule dimerizer.

[0214] A composition of the present disclosure can comprise, in addition to the synthetic immune cell and the second immune cell, a pharmaceutically acceptable excipient. Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985.

Methods

[0215] The present disclosure provides a method of increasing proliferation and/or activity of a target immune cell in an individual. The method comprises administering to an individual in need thereof an effective amount (number) of a synthetic immune cell of the present disclosure, or an effective amount of a composition of the present disclosure (where the composition comprises a synthetic immune cell of the present disclosure and a second immune cell).

[0216] The target immune cell in the individual can be a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a natural killer (NK) cell, or a regulatory T cell (Treg). The target immune cell in the individual can be one that is not genetically modified. The target immune cell in the individual can be one that is genetically modified, e.g., genetically modified to express an exogenous TCR, a CAR, or a BiTE. The target immune cell can be one that has been genetically modified to express a variant IL-2R.beta. polypeptide on its cell surface. For example, the target immune cell can be one that has been genetically modified to express a variant IL-2R.beta. polypeptide, as described above, where the variant IL-2R.beta. binds an ortho-IL-2 polypeptide as described above.

[0217] In some cases, the target immune cell is specific for a cancer-associated antigen. In some cases, the target immune is specific for the same cancer-associated antigen to which the antibody present in the chimeric polypeptide binds. In some cases, the target immune cell is specific for a cancer-associated antigen; and the antibody present in the chimeric polypeptide is specific for a tissue-specific antigen. In some cases, the target immune cell is specific for a cancer-associated antigen; and the antibody present in the chimeric polypeptide is specific for a cell type-specific antigen.

[0218] In some cases, a method of the present disclosure comprises administering to an individual in need thereof a synthetic immune cell of the present disclosure, where from 10.sup.2 to 10.sup.9 synthetic immune cells of the present disclosure are administered. For example, from 10.sup.2 to about 10.sup.3, from about 10.sup.3 to about 10.sup.4, from about 10.sup.4 to about 10.sup.5, from about 10.sup.5 to about 10.sup.6, from about 10.sup.6 to about 10.sup.7, from about 10.sup.7 to about 10.sup.8, or from about 10.sup.8 to about 10.sup.9, synthetic immune cells of the present disclosure are administered.

[0219] In some cases, a method of the present disclosure comprises administering to an individual in need thereof: a) a synthetic immune cell of the present disclosure; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of a target effector immune cell. Suitable cytokines are described above. In some cases, the cytokine is a variant IL-2 polypeptide, as described above.

[0220] In some instances, a method of the instant disclosure finds use in treating a cancer. Cancers that can be treated using a method of the present disclosure include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer (Extrahepatic), Bladder Cancer, Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid/Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc.), Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Duct (e.g., Bile Duct, Extrahepatic, etc.), Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (e.g., Intraocular Melanoma, Retinoblastoma, etc.), Fibrous Histiocytoma of Bone (e.g., Malignant, Osteosarcoma, ect.), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor (e.g., Extracranial, Extragonadal, Ovarian, Testicular, etc.), Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis (e.g., Langerhans Cell, etc.), Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kaposi Sarcoma, Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (e.g., Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, etc.), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer (e.g., Non-Small Cell, Small Cell, etc.), Lymphoma (e.g., AIDS-Related, Burkitt, Cutaneous T-Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.), Macroglobulinemia (e.g., Waldenstrom, etc.), Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g., Chronic (CML), etc.), Myeloid Leukemia (e.g., Acute (AML), etc.), Myeloproliferative Neoplasms (e.g., Chronic, etc.), Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer (e.g., Lip, etc.), Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (e.g., Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, etc.), Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, and the like. Thus, in some cases, an individual in need thereof is an individual who has a cancer, e.g., who has been diagnosed as having a cancer.

[0221] In some cases, a method of the present disclosure finds use in treating an autoimmune disorder. Thus, in some cases, an individual in need thereof is an individual who has been diagnosed as having an autoimmune disorder.

Examples of Non-Limiting Aspects of the Disclosure

[0222] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-55 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0223] Aspect 1. A genetically modified, in vitro immune cell, wherein the immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, wherein the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator.

[0224] Aspect 2. The genetically modified immune cell of aspect 1, wherein the immune cell is a T cell or a macrophage.

[0225] Aspect 3. The genetically modified immune cell of aspect 1, wherein the binding triggered transcriptional activator comprises, from N-terminal to C-terminal and in covalent linkage: [0226] i) an extracellular domain comprising an antibody specific for a target antigen; [0227] ii) a Notch regulatory polypeptide that comprises one or more proteolytic cleavage sites; and [0228] iii) an intracellular domain comprising a transcriptional activator,

[0229] wherein binding of the antibody to the target antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain.

[0230] Aspect 4. The genetically modified immune cell of aspect 3, wherein the Notch regulatory region comprises a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site.

[0231] Aspect The genetically modified immune cell of aspect 3 or aspect 4, wherein the Notch regulatory region further comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats.

[0232] Aspect 6. The genetically modified immune cell of aspect 1, wherein the binding triggered transcriptional activator comprises, from N-terminal to C-terminal and in covalent linkage: [0233] a) an extracellular domain comprising an antibody specific for a target antigen; [0234] b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site; [0235] c) a cleavable transmembrane domain; and [0236] d) an intracellular domain comprising a Notch intracellular signaling domain comprising a transcriptional activator, wherein binding of the antibody to the target antigen induces cleavage of the non-Notch force sensor cleavage domain at the proteolytic cleavage site, thereby releasing the intracellular domain, and wherein the non-Notch force sensor cleavage domain is selected from the group consisting of: a von Willebrand Factor (vWF) cleavage domain, an amyloid-beta cleavage domain, a CD16 cleavage domain, a CD44 cleavage domain, a Delta cleavage domain, a cadherin cleavage domain, an ephrin-type receptor or ephrin ligand cleavage domain, a protocadherin cleavage domain, a filamin cleavage domain, a synthetic E cadherin cleavage domain, an interleukin-1 receptor type 2 (IL1R2) cleavage domain, a major prion protein (PrP) cleavage domain, a neuregulin cleavage domain and an adhesion-GPCR cleavage domain,

[0237] Aspect 7. The genetically modified immune cell of aspect 6, wherein the non-Notch force sensor cleavage domain is a vWF cleavage domain.

[0238] Aspect 8. The genetically modified immune cell of aspect 7, wherein the vWF cleavage domain comprises a vWF A2 domain or a variant thereof.

[0239] Aspect 9. The genetically modified immune cell of any one of aspects 1-8, wherein the antibody is a nanobody, a diabody, a triabody, a minibody, a F(ab').sub.2 fragment, a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody (sdAb).

[0240] Aspect 10. The genetically modified immune cell of any one of aspects 1-9, wherein the cytokine is IL-2.

[0241] Aspect 11. The genetically modified immune cell of aspect 10, wherein the IL-2 is an IL-2 variant that exhibits increased binding affinity for IL-2R13 compared to wild-type IL-2.

[0242] Aspect 12. The genetically modified T cell of aspect 11, wherein the IL-2 variant comprises amino acid substitutions L80F, R81D, L85V, I86V, and I92F, compared to wild-type human IL-2.

[0243] Aspect 13. The genetically modified immune cell of aspect 10, wherein the IL-2 is a variant that preferentially activates regulatory T cells (T regs).

[0244] Aspect 14. The genetically modified immune cell of aspect 10, wherein the IL-2 is a variant that preferentially activates natural killer (NK) cells.

[0245] Aspect 15. The genetically modified immune cell of aspect 10, wherein the IL-2 is: [0246] i) a variant IL-2 that binds to a variant IL-2R.beta. comprising one or more amino acid substitutions selected from Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R; and ii) exhibits reduced binding to wild-type IL-2R.beta..

[0247] Aspect 16. The genetically modified immune cell of aspect 15, wherein the variant IL-2 comprises one or more amino acid substitutions selected from: i) H16N, L19V, D20N, Q22T, M23H, and G27K; ii) E15D, H16N, L19V, D20L, Q22T, and M23H; iii) E15D, H16N, L19V, D20L, Q22T, and M23A; or iv) E15D, H16N, L19V, D20L, Q22K, M23A.

[0248] Aspect 17. The genetically modified immune cell of any one of aspects 1-9, wherein the cytokine is IL-15 or IL7.

[0249] Aspect 18. The genetically modified immune cell of any one of aspects 1-9, wherein the proliferation-inducing polypeptide binds IL-2R.beta..gamma..sub.c heterodimer, but does not bind IL-2R.alpha. or IL-2R.beta..

[0250] Aspect 19. The genetically modified immune cell of any one of aspects 1-18, wherein the target antigen is a cancer-associated antigen.

[0251] Aspect 20. The genetically modified immune cell of aspect 19, wherein the cancer-associated antigen is selected from CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, and GD2.

[0252] Aspect 21. The genetically modified immune cell of any one of aspects 1-18, wherein the target antigen is tissue-specific antigen or an organ-specific antigen or a cell type-specific antigen.

[0253] Aspect 22. The genetically modified immune cell of any one of aspects 1-18, wherein the target antigen is a stromal cell antigen.

[0254] Aspect 23. The genetically modified immune cell of any one of aspects 1-22, wherein the genetically modified immune cell is a T cell that does not express an endogenous major histocompatibility complex (MHC) class I polypeptide on its surface.

[0255] Aspect 24. The genetically modified immune cell of aspect 23, wherein the genetically modified immune cell comprises a deletion of all or a portion of at least one MHC class I coding region.

[0256] Aspect 25. The genetically modified immune cell of aspect 24, wherein the at least one MHC class I coding region is a .beta.2-microglobulin coding region.

[0257] Aspect 26. The genetically modified immune cell of any one of aspects 1-25, wherein the genetically modified immune cell is a T cell that does not express an endogenous T-cell receptor (TCR).

[0258] Aspect 27. A composition comprising: [0259] a) the genetically modified immune cell of any one of aspects 1-26; and [0260] b) a cytotoxic T cell (CTL).

[0261] Aspect 28. The composition of aspect 27, wherein the CTL is genetically modified to express: [0262] a) an exogenous T-cell receptor (TCR), wherein the exogenous TCR is specific for a target antigen; or [0263] b) a chimeric antigen receptor (CAR), wherein the CAR is specific for a target antigen; or [0264] c) a bispecific T-cell engager (BiTE), wherein the BiTE comprises: i) a first antigen-binding region specific for CD3; and ii) a second antigen-binding region specific for a target antigen other than CD3.

[0265] Aspect 29. The composition of aspect 27 or aspect 28, wherein the TCR, the CAR, or the BiTE is specific for a cancer-associated antigen, and wherein the antibody present in the chimeric polypeptide is specific for a cancer-associated antigen.

[0266] Aspect 30. The composition of aspect 29, wherein the TCR, the CAR, or the BiTE is specific for the same cancer-associated antigen as the cancer-associate antigen to which the antibody present in the chimeric polypeptide binds.

[0267] Aspect 31. The composition of aspect 29, wherein the TCR, the CAR, or the BiTE is specific for a cancer-associated antigen that is different from the cancer-associate antigen to which the antibody present in the chimeric polypeptide binds.

[0268] Aspect 32. The composition of any one of aspects 28-31, wherein the CTL is genetically modified to express a CAR, and wherein the CAR comprises: a) an extracellular domain comprising the antigen-binding domain; b) a transmembrane region; and c) an intracellular signaling domain.

[0269] Aspect 33. The composition of aspect 32, wherein the intracellular signaling domain comprises: i) a signaling domain from the zeta chain of human CD3; and ii) one or more costimulatory polypeptides.

[0270] Aspect 34. The composition of aspect 33, wherein the one or more costimulatory polypeptides is selected from CD28, 4-1BB, and OX-40.

[0271] Aspect 35. The composition of any one of aspects 32-34, wherein the CAR is a single polypeptide chain.

[0272] Aspect 36. The composition of any one of aspects 32-34, wherein the CAR comprises 2 polypeptide chains.

[0273] Aspect 37. The composition of aspect 36, wherein the 2 polypeptide chains dimerize in the presence of a small molecule dimerizer.

[0274] Aspect 38. A method of increasing proliferation and/or activity of a target immune cell in an individual, the method comprising administering to the individual a genetically modified immune cell according to any one of aspects 1-26 or a composition according to any one of aspects 27-37.

[0275] Aspect 39. The method of any one of aspects 38-40, wherein the target immune cell is a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a natural killer (NK) cell, or a regulatory T cell (Treg).

[0276] Aspect 40. The method of aspect 38 or aspect 39, wherein the target immune cell is an endogenous immune cell.

[0277] Aspect 41. The method of aspect 38 or aspect 39, wherein the target immune cell is an exogenous immune cell that has been genetically modified and introduced into the individual.

[0278] Aspect 42. The method of aspect 41, wherein the target immune cell is T cell that has been genetically modified to express an exogenous T-cell receptor (TCR) or an exogenous chimeric antigen receptor (CAR).

[0279] Aspect 43. The method of aspect 41 or aspect 42, wherein the target immune cell is genetically modified to express a variant IL2 receptor on its surface.

[0280] Aspect 44. The method of any one of aspects 38-43, wherein the target immune cell is specific for a cancer-associated antigen.

[0281] Aspect 45. The method of aspect 44, wherein the target immune cell is specific for the same cancer-associated antigen to which the antibody present in the chimeric polypeptide binds.

[0282] Aspect 46. The method of any one of aspects 38-45, comprising administering to the individual an effective amount of a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell.

[0283] Aspect 47. The method of aspect 46, wherein the cytokine is IL-2.

[0284] Aspect 48. The method of aspect 47, wherein the IL-2 is an IL-2 variant that exhibits increased binding affinity for IL-2R13 compared to wild-type IL-2.

[0285] Aspect 49. The method of aspect 48, wherein the IL-2 variant comprises amino acid substitutions L80F, R81D, L85V, I86V, and I92F, compared to wild-type human IL-2.

[0286] Aspect 50. The method of aspect 47, wherein the IL-2 is a variant that preferentially activates regulatory T cells (T regs).

[0287] Aspect 51. The method of aspect 47, wherein the IL-2 is a variant that preferentially activates natural killer (NK) cells.

[0288] Aspect 52. The method of aspect 47, wherein the IL-2 is: i) a variant IL-2 that binds to a variant IL-2R.beta. comprising one or more amino acid substitutions selected from Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R; and ii) exhibits reduced binding to wild-type IL-2R.beta..

[0289] Aspect 53. The method of aspect 52, wherein the variant IL-2 comprises one or more amino acid substitutions selected from: i) H16N, L19V, D2ON, Q22T, M23H, and G27K; ii) E15D, H16N, L19V, D20L, Q22T, and M23H; iii) E15D, H16N, L19V, D20L, Q22T, and M23A; or iv) E15D, H16N, L19V, D20L, Q22K, M23A.

[0290] Aspect 54. The method of aspect 46, wherein the cytokine is IL-15 or IL-7.

[0291] Aspect 55. The method of aspect 46, wherein the proliferation-inducing polypeptide binds IL-2R.beta..gamma..sub.c heterodimer, but does not bind IL-2R.alpha. or IL-2R.beta..

EXAMPLES

[0292] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1

[0293] Synthetic helper T cells were designed and generated. The synthetic helper T cells inducibly secrete proliferative cytokines upon recognition of a local tumor antigen. These synthetic helper cells expanded effector T cells or NK cells by secreting an enhanced-affinity interleukin (IL)-2. In a two-tumor mouse model, synthetic helper cells locally expanded T cells only in a tumor expressing the cognate triggering antigen, and potently enhanced tumor killing by T cells expressing a weak TCR.

Materials and Methods

Lentiviral DNA Constructs

[0294] SynNotch sequences such as "anti-CD19 synNotch" (anti-CD19-scFv-Notch-Gal4VP64) or "anti-GFP synNotch (LaG16-LaG2 tandem anti-GFP nanobody-Notch-Gal4VP64) were cloned downstream of a pGK or SFFV promoter, and response elements contained payloads (typically fluorescent protein-P2A-cytokine) were cloned downstream of a mCMV promoter and GAL4UAS, both in the pHR backbone, either a single multicistronic constructs or one for each component.

T Cell Culture

[0295] T cells were isolated from Leukopaks using CD8+ selection kits, following which they were frozen in RPMI with 20% human AB serum and 10% DMSO. For assays, frozen T cells were thawed in T cell media (ImmunoCult) with IL-2 (always 30 U/mL unless otherwise specified) and resuspended at 1e6 cells/mL on day -13 (relative to the experiment start date) and activated with 25 uL anti-CD3/anti-CD28 beads per 1e6 T cells on day -12. Meanwhile, Lx293t lentiviral packaging cells were seeded in 6-well plates at 7e5 cells/well in DMEM F21 with 10% FBS on day -14, and transfected with pHR constructs for transfer into T cells as well as pCMV and pMD2.g packaging plasmids using FuGene on day -13. On day -11, viral supernatant was collected, cells removed by centrifugation at 500.times.g for 5 min, and applied to T cells at 1 mL per le7 T cells for single construct transductions or 0.75 mL of each construct for double construct transductions in 24-well plates. On day -10, T cells with virus were centrifuged at 400.times.g for 4 minutes, virus discarded, and T cells resuspended in media with IL-2. Cells were sorted for positive transduction on a BD FACSAria II or FACSAria Fusion on day -7. Cells were sorted for expression of a fluorescent protein marker or for positive staining of a Myctag on synNotch, or both.

[0296] Cells with synNotch were also negatively sorted on the fluorescent protein marker of synNotch activation in order to remove cells with high basal activity of synNotch (leaky cells). On days -5 through -3, T cells were counted with a Countess and Immunocult with IL-2 was added to dilute cell concentration to 5e5 cells/mL. On day 0, cells were used in assays in vitro or in vivo assays.

Tumor Cell Culture

[0297] K562 cells were cultured in Iscove's Modified Dulbecco's Modified Eagle Medium with 10% FBS and split to 2.5e5 cells/mL every 3 days or 3.5e5 cells/mL every 2 days. A375 cells were cultured in DMEM with Glutamax and 10% FBS and split 1:6 every 2 days or 1:10 every 3 days.

In Vitro Assay Preparation

[0298] T cells and target cells were washed of residual media and cytokines by two rounds of centrifugations at 400.times.g for 4 minutes followed by resuspension in Immunocult without IL-2. In some cases, T cells were stained with 1:5000 CellTrace proliferation stain (source) following manufacturer's protocol. After counting, various numbers of T cells and tumor cells were added to wells of 96 well plates, with or without various concentrations of IL-2, in 200 uL total volumes, with 2-3 replicates per conditions. For continuously-cultured experiments, at least 25 uL of each well was removed for analysis at regular time intervals (typically every 2-4 days) and replaced with fresh media. For closed-end experiments, multiple replicate time points were created on day 0 and analyzed destructively at different days (typically every 2-4 days).

Mouse Experiments

[0299] T cells were washed in phosphate-buffered saline (PBS), resuspended at 10 times the injection amount per mL, and 100 uL was injected into immunocompromised NOD scid gamma mice via the tail vein on day 0. Target tumor cells washed in PBS, resuspend at 10 times the injection amount per mL, and 100 uL was injected subcutaneously in the flank on day 0 (experiments in FIG. 3) or day -4 (experiments in FIG. 4). Tumor size was measured by caliper. Bioluminescence imaging was performed using an IVIS as follows: mice were injected intraperitoneally with 200 uL d-luciferin and imaged 15 minutes later. Excised tumors were cut in half and send for histology by HistoWiz or dissociated to perform flow cytometry. To prepare for flow cytometry, tumors were minced with razor blades, digested with collagenase and DNAse, passed through cell strainers, washed with PBS with EDTA, and stained. Excised spleens were prepared for flow cytometry by gently forcing through cell strainers with a rubber syringe plunger, then washing with PBS.

Flow Cytometry and Analysis

[0300] Cells in 96 well plates were analyzed by flow cytometry on a BD LSRii or BD LSRFortessa X-20 with high-throughput system. For cell counting experiments, cells were not washed to ensure minimal errors due to cell loss: cells in media were mixed and transferred from the assay plates to new round bottom plates and PBS with Sytox Red, Blue, or Green was added to a final dilution of 1:500 in 200 uL total volume. For other experiments, plates were centrifuged at 400.times.g for 4 minutes, supernatant discarded, and stained with 1:500 Live/Dead Near Infrared in PBS. Plates were centrifuged, supernatant discarded, followed by staining with fluorescently labeled antibodies at various dilutions in PBS with 5% FBS. Wells were washed twice with and resuspended in PBS with 5% FBS. Compensation was performed using single antibody-stained Ultracomp beads, single antibody-stained transduced or untransduced cells, or unstained transduced cells expressing a single fluorescent protein. Analysis of flow data was performed in FlowJo. Cells were resolved from debris, anti-myctac beads, and bubbles based on forward scatter and side scatter signal. Cell singlets were isolated by the ratio of forward scatter area to height signal. Live cells were discriminated using Sytox or Live/Dead signal. Different cell types were distinguished based on the presence or absence unique fluorescent protein expression or antibody staining combinations.

Results

[0301] The results are shown in FIGS. 1-6.

[0302] FIG. 1A-1C: A. Controlling immune cell expansion is critical for cell therapies to balance between amplification and side effects. B. Local proliferation is important to enhance therapies at relevant tissues without causing systemic toxicity. C. A synthetic helper T cell could recognize target tissues or tumors and secrete cytokines to cause local proliferation of immune cells.

[0303] FIG. 2A-2C: Synthetic helper T cells with inducible cytokine circuits for immune cell expansion. A. Synthetic helper T cells use synNotch to secrete cytokines in response to target cell recognition, which are sensed via endogenous receptors, increasing survival, growth, and activation. B. Synthetic helper T cells were co-cultured with CD8+ effector T cells and stimulated with anti-myctag beads that trigger synNotch by binding the myc-tag on its N-terminus. Both synthetic helper T cells (left below diagram, blue) and effector T cells (right below diagram, orange) proliferated in response to stimulating beads but died in response to control beads (dark vs. light color). Effector cells proliferated more than synthetic helper cells (dashed blue line). CFSE dilution confirms fold change data (right of diagram): synthetic helper cells inducibly caused effector proliferation comparable to high doses of IL-2 (increasing from light gray to black: 0, 30, 100, 300 U/mL). C. Synthetic helper T cells were co-cultured with NK cells and K562 tumor cells expressing CD19 ligand (dark lines) or not (light lines). SynNotch binding of CD19 caused synthetic helper cells to secrete Super-2, which caused T cell and NK cell proliferation (right column, top and bottom respectively) and killing of K562 cells (lower left).

[0304] FIG. 3A-3E. Synthetic helper T cells can drive locally targeted proliferation in vivo. A. Setup of mouse experiment with bilateral tumors. Right ("target") tumors express CD19 ligand whereas left ("off-target") tumors do not. Synthetic helper T cells with anti-CD19 synNotch driving Super-2 were coinjected with effector T cells expressing eff-luc. B. Bioluminescence imaging of eff-luc expressing effector T cells with or without helpers over 18 days following T cell injection. Orange circles highlight proliferation in the target tumor. One characteristic mouse per group is shown. C. Quantification of bioluminescence signal in each tumor for mice receiving both effector and synthetic helper T cells showing specific effector T cell expansion in the target tumor. D. Ratio of luminescence signal in the target tumor to that in the off-target tumor, showing that effector cells with synthetic helper cells preferentially localize on the target side, whereas effector cells alone have no target/off-target localization preference. D. Tumors were harvested at day 21 from mice given both effector and synthetic helper cells and dissociated. Flow cytometry resolves CD8+ T cells from non-T cells and tagBFP+synthetic helper cells from effector cells. mCherry, a marker of synNotch activation, was more frequently expressed among synthetic helper cells in the target tumor than the off-target tumor. E. Percentage of T cells (among all live cell events in flow analysis) and percentage of effectors (among T cell events) show higher frequency in the target tumor relative to the off-target tumor.

[0305] FIG. 4A-4C. Bioluminescence images of individual mice over 18 days, from the same experiment shown in FIG. 3A-3E, showing accumulation of effluc+effector T cells in the target tumor (orange circle) only when synthetic helper T cells are coinjected (right group under blue line). Without synthetic helper T cells (left group under orange line), effector cells tend to localize to the spleen in small amounts but do not accumulate in either tumor. B. Time trajectories of total bioluminescence signal from effluc+effector T cell in either the target (blue or orange line) or off-target tumor (gray), quantified from the images in A, confirming specific accumulation of effector T cells in the target tumor when synthetic helper T cells are added (top row as compared to bottom). C. Replication of the experiment in FIG. 3A-3E with T cells from another donor (top left: group averages; top right: individual mice) as well as an additional run with larger tumors created by injecting 5e6 K562s rather of 1e6 (bottom left: group averages; bottom right: individual mice), both confirming the same trend on average.

[0306] FIG. 5A-5D. A. Synthetic helpers form a functional AND-gate circuit with cytotoxic effectors to specifically enhance local killing of target tumors. B. In vivo experiment in NSG mice C. Tumor volume increases for effectors only but decreases in a target-specific way when synthetic helpers are added. D. Bioluminescence of effectors at an early time point (day 8) shows that synthetic helpers caused proliferation specifically in the target tumor.

[0307] FIG. 6A-6B. Tumor volume trajectories of individual mice from the experiment shown in FIG. 5A-5D. Lower target tumor volume (top 5 plots, gray) vs. off-target (blue) demonstrates specific killing of the target tumor by anti-NY-ESO TCR+effector T cells only in the presence of coinjected synthetic helper T cells. Without synthetic helper T cells (bottom 5 plots), anti-NY-ESO TCR+effector T cells are ineffective at reducing the volume of either target (gray) or off-target (purple) tumors. B. Replication of the experiment shown in FIG. 5A-5D with T cells from another donor. Average tumor volume over time (left two plots) as well as tumor volume trajectories for individual mice (right 10 plots, following the layout of A) confirm the same trend.

[0308] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Sequence CWU 1

1

1141153PRTHomo sapiens 1Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu65 70 75 80Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140Cys Gln Ser Ile Ile Ser Thr Leu Thr145 1502133PRTArtificial sequencesynthetic sequence 2Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr 1303525PRTHomo sapiens 3Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala1 5 10 15Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu65 70 75 80Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu145 150 155 160Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn225 230 235 240Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro305 310 315 320Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp385 390 395 400Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg465 470 475 480Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 515 520 5254154PRTArtificial sequencesynthetic sequence 4Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu65 70 75 80Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95Asn Phe His Phe Asp Pro Arg Asp Val Val Ile Ser Asn Ile Asn Val 100 105 110Phe Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr 115 120 125Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr 130 135 140Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr145 1505134PRTArtificial sequencesynthetic sequence 5Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Ile Asn Val Phe Val Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 1306134PRTArtificial sequencesynthetic sequence 6Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Val Asn Val Phe Val Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 1307134PRTArtificial sequencesynthetic sequence 7Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Val Asn Val Phe Ile Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 1308134PRTArtificial sequencesynthetic sequence 8Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala His Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Ile Asn Val Phe Val Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 1309134PRTArtificial sequencesynthetic sequence 9Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Ser Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Ile Asn Val Phe Val Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 13010134PRTArtificial sequencesynthetic sequence 10Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Asn Ser Lys Asn Phe His Phe65 70 75 80Asp Pro Arg Asp Val Val Ile Ser Asn Ile Asn Val Phe Val Leu Glu 85 90 95Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125Ile Ile Ser Thr Leu Thr 13011133PRTArtificial sequencesynthetic sequence 11Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Ser Gln1 5 10 15Leu Leu Val Thr Leu Lys Leu Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr 13012525PRTArtificial sequencesynthetic sequence 12Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala1 5 10 15Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60Gly Ala Pro Asp Ser Tyr Lys Leu Asp Thr Val Asp Ile Val Thr Leu65 70 75 80Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125Ser Gln Ala Ser Asp Phe Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu145 150 155 160Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu Gly His Leu Leu Val Gly 210 215 220Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn225 230 235 240Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr 245 250 255Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly 260 265 270Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser 275 280 285Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg 290 295 300Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro305 310 315 320Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln 325 330 335Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys 340 345 350Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu 355 360 365Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro 370 375 380Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp385 390 395 400Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser 405 410 415Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser 420 425 430Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro 435 440 445Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu 450 455 460Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg465 470 475 480Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe 485 490 495Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser 500 505 510Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val 515 520

5251356PRTArtificial sequencesynthetic sequence 13Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val Glu Pro Pro1 5 10 15Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 30Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg Arg 35 40 45Arg Gln Leu Cys Ile Gln Lys Leu 50 551456PRTArtificial sequencesynthetic sequence 14Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val Glu Pro Pro1 5 10 15Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 30Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg Arg 35 40 45Arg Gln Leu Cys Ile Gln Lys Leu 50 5515120PRTArtificial sequencesynthetic sequence 15Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp1 5 10 15Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly 20 25 30Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 35 40 45Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 50 55 60Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys65 70 75 80Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 85 90 95Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu 100 105 110Cys Glu Trp Asp Gly Leu Asp Cys 115 12016152PRTArtificial sequencesynthetic sequence 16Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu1 5 10 15Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 20 25 30Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 35 40 45Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 50 55 60Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly65 70 75 80Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 85 90 95Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 100 105 110Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 115 120 125Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser 130 135 140Glu Pro Val Glu Pro Pro Leu Pro145 1501723PRTArtificial sequencesynthetic sequence 17His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val1 5 10 15Gly Cys Gly Val Leu Leu Ser 2018306PRTArtificial sequencesynthetic sequence 18Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp1 5 10 15Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly 20 25 30Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 35 40 45Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 50 55 60Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys65 70 75 80Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 85 90 95Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu 100 105 110Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 115 120 125Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 130 135 140Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His145 150 155 160Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 165 170 175Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 180 185 190Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 195 200 205Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 210 215 220Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln225 230 235 240Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 245 250 255Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser 260 265 270Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 275 280 285Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 290 295 300Leu Ser30519358PRTArtificial sequencesynthetic sequence 19Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro1 5 10 15Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn 20 25 30Gly Leu Leu Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly 35 40 45Arg Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu 50 55 60Cys Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn65 70 75 80His Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp 85 90 95Pro Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser 100 105 110Asp Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp 115 120 125Gly Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp 130 135 140Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys145 150 155 160Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val 165 170 175Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro 180 185 190Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser 195 200 205His Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln 210 215 220Gln Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His225 230 235 240Pro Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro 245 250 255Gly Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile 260 265 270Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln 275 280 285Ser Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu 290 295 300Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu305 310 315 320Ala Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His 325 330 335Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 340 345 350Cys Gly Val Leu Leu Ser 3552037PRTArtificial sequencesynthetic sequence 20Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro1 5 10 15Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn 20 25 30Gly Leu Leu Cys His 352137PRTArtificial sequencesynthetic sequence 21Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg His Gly Ala Ser Cys1 5 10 15Gln Asn Thr His Gly Gly Tyr Arg Cys His Cys Gln Ala Gly Tyr Ser 20 25 30Gly Arg Asn Cys Glu 352237PRTArtificial sequencesynthetic sequence 22Asp Ile Asp Asp Cys Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys1 5 10 15Thr Asp Gly Ile Asn Thr Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg 20 25 30Gly Thr Phe Cys Glu 352337PRTArtificial sequencesynthetic sequence 23Asp Val Asn Glu Cys Asp Ser Gln Pro Cys Leu His Gly Gly Thr Cys1 5 10 15Gln Asp Gly Cys Gly Ser Tyr Arg Cys Thr Cys Pro Gln Gly Tyr Thr 20 25 30Gly Pro Asn Cys Gln 352436PRTArtificial sequencesynthetic sequence 24Leu Val Asp Glu Cys Ser Pro Ser Pro Cys Gln Asn Gly Ala Thr Cys1 5 10 15Thr Asp Tyr Leu Gly Gly Tyr Ser Cys Lys Cys Val Ala Gly Tyr His 20 25 30Gly Val Asn Cys 352536PRTArtificial sequencesynthetic sequence 25Ile Asp Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu1 5 10 15Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly 20 25 30Val His Cys Glu 352627PRTArtificial sequencesynthetic sequence 26Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val Gly Gly Tyr Ser Cys1 5 10 15Thr Cys Pro Pro Gly Phe Val Gly Glu Arg Cys 20 252710PRTArtificial sequencesynthetic sequence 27Gly Arg Arg Arg Arg Glu Leu Asp Pro Met1 5 10289PRTArtificial sequencesynthetic sequence 28Arg Gln Arg Arg Glu Leu Asp Pro Met1 5298PRTArtificial sequencesynthetic sequence 29Lys Ile Glu Ala Val Lys Ser Glu1 5308PRTArtificial sequencesynthetic sequence 30Lys Ile Glu Ala Val Gln Ser Glu1 5318PRTArtificial sequencesynthetic sequence 31Val Gly Cys Gly Val Leu Leu Ser1 5327PRTArtificial sequencesynthetic sequence 32Gly Cys Gly Val Leu Leu Ser1 533248PRTArtificial sequencesynthetic sequence 33Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu1 5 10 15Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg65 70 75 80Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr145 150 155 160Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp225 230 235 240Phe Asp Leu Asp Met Leu Pro Gly 24534211PRTArtificial sequencesynthetic sequence 34Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu1 5 10 15Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile65 70 75 80Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140Thr Val Ser Ala Ala Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp145 150 155 160Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp 165 170 175Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp 180 185 190Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met 195 200 205Leu Gly Ser 21035120PRTArtificial sequencesynthetic sequence 35Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp1 5 10 15Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly 20 25 30Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 35 40 45Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 50 55 60Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys65 70 75 80Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 85 90 95Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu 100 105 110Cys Glu Trp Asp Gly Leu Asp Cys 115 12036152PRTArtificial sequencesynthetic sequence 36Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu1 5 10 15Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 20 25 30Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 35 40 45Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 50 55 60Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly65 70 75 80Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 85 90 95Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 100 105 110Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 115 120 125Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser 130 135 140Glu Pro Val Glu Pro Pro Leu Pro145 150374PRTArtificial sequencesynthetic sequence 37Arg Gln Arg Arg13823PRTArtificial sequencesynthetic sequence 38His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val1 5 10 15Gly Cys Gly Val Leu Leu Ser 20394PRTArtificial sequencesynthetic sequence 39Val Leu Leu Ser14037PRTArtificial sequencesynthetic sequence 40Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro1 5 10 15Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn 20 25 30Gly Leu Leu Cys His 354156PRTArtificial sequencesynthetic sequence 41Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val Glu Pro Pro1 5 10 15Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 30Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg Arg 35 40 45Arg Gln Leu Cys Ile Gln Lys Leu 50 5542206PRTArtificial sequencesynthetic sequence 42Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65

70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly 195 200 20543248PRTArtificial sequencesynthetic sequence 43Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu1 5 10 15Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg65 70 75 80Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr145 150 155 160Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp225 230 235 240Phe Asp Leu Asp Met Leu Pro Gly 24544273PRTArtificial sequencesynthetic sequence 44Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala1 5 10 15Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu 20 25 30Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu 35 40 45Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu 50 55 60Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln65 70 75 80Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn 85 90 95Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly 100 105 110Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser 115 120 125Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp 130 135 140Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp145 150 155 160Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser 165 170 175Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly 180 185 190Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu 195 200 205Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met 210 215 220Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys225 230 235 240Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile 245 250 255Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp 260 265 270Lys45242PRTArtificial sequencesynthetic sequence 45Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser46242PRTArtificial sequencesynthetic sequence 46Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser47244PRTArtificial sequencesynthetic sequence 47Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro1 5 10 15Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 35 40 45Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln 50 55 60Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr65 70 75 80Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser145 150 155 160Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly 165 170 175Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 180 185 190Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu 195 200 205Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln 210 215 220Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu225 230 235 240Ile Lys Ala Ser48261PRTArtificial sequencesynthetic sequence 48Gly Ser Gln Val Gln Leu Gln Gln Gln Val Gln Leu Gln Glu Ser Gly1 5 10 15Gly Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala 20 25 30Ser Gly Phe Thr Phe Ser His Tyr Gly Met Ser Trp Val Arg Gln Thr 35 40 45Pro Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Gly Ser Arg Gly Thr 50 55 60Tyr Thr His Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg65 70 75 80Asp Asn Asp Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser 85 90 95Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Ser Glu Phe Tyr Tyr 100 105 110Tyr Gly Asn Thr Tyr Tyr Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly 115 120 125Ala Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Phe145 150 155 160Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 165 170 175Glu Ser Val Asp Asn Tyr Gly Phe Ser Phe Met Asn Trp Phe Gln Gln 180 185 190Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ile Ser Asn Arg 195 200 205Gly Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220Phe Ser Leu Asn Ile His Pro Val Glu Glu Asp Asp Pro Ala Met Tyr225 230 235 240Phe Cys Gln Gln Thr Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255Lys Leu Glu Ile Lys 26049127PRTArtificial sequencesynthetic sequence 49Met Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30Thr Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ala Arg Ile Thr Trp Ser Ala Gly Tyr Thr Ala Tyr Ser Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Ser Arg Ser Ala Gly Tyr Ser Ser Ser Leu Thr Arg Arg 100 105 110Glu Asp Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 12550128PRTArtificial sequencesynthetic sequence 50Met Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ile Ser 20 25 30Met Ala Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ala Gly Ile Ser Arg Ser Ala Gly Ser Ala Val His Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Val Arg Thr Ser Gly Phe Phe Gly Ser Ile Pro Arg Thr 100 105 110Gly Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 12551128PRTArtificial sequencesynthetic sequence 51Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr1 5 10 15Gly Gly Ser Leu Lys Leu Ser Cys Thr Ala Ser Val Arg Thr Leu Ser 20 25 30Tyr Tyr His Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ala Gly Ile His Arg Ser Gly Glu Ser Thr Phe Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Val His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Gln Arg Val Arg Gly Phe Phe Gly Pro Leu Arg Ser Thr 100 105 110Pro Ser Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 12552270PRTArtificial sequencesynthetic sequence 52Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala1 5 10 15Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu 35 40 45Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg Ala Lys Asn Thr65 70 75 80Val Asp Leu Gln Met Asp Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ser Ala Arg Ser Arg Gly Tyr Val Leu Ser Val Leu Arg Ser 100 105 110Val Asp Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met 130 135 140Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly145 150 155 160Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn 165 170 175Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 180 185 190Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp Ser 195 200 205Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val 210 215 220Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr225 230 235 240Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg Val 245 250 255Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 260 265 27053242PRTArtificial sequencesynthetic sequence 53Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105

110Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg145 150 155 160Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val225 230 235 240Ser Ser54211PRTArtificial sequencesynthetic sequence 54Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu1 5 10 15Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile65 70 75 80Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140Thr Val Ser Ala Ala Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp145 150 155 160Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp 165 170 175Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp 180 185 190Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met 195 200 205Leu Gly Ser 21055205PRTArtificial sequencesynthetic sequence 55Leu Glu Ile Arg Ala Ala Phe Leu Arg Gln Arg Asn Thr Ala Leu Arg1 5 10 15Thr Glu Val Ala Glu Leu Glu Gln Glu Val Gln Arg Leu Glu Asn Glu 20 25 30Val Ser Gln Tyr Glu Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys Gly 35 40 45Gly Ser Gly Gly Ser Gly Gly Ser Met Lys Leu Leu Ser Ser Ile Glu 50 55 60Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser Lys Glu65 70 75 80Lys Pro Lys Cys Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr 85 90 95Ser Pro Lys Thr Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu 100 105 110Val Glu Ser Arg Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe 115 120 125Pro Arg Glu Asp Leu Asp Met Ile Leu Lys Met Asp Ser Leu Gln Asp 130 135 140Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys145 150 155 160Asp Ala Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu 165 170 175Thr Leu Arg Gln His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser 180 185 190Ser Asn Lys Gly Gln Arg Gln Leu Thr Val Ser Ala Ala 195 200 20556185PRTArtificial sequencesynthetic sequence 56Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro1 5 10 15Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 35 40 45Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln 50 55 60Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr65 70 75 80Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser145 150 155 160Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly 165 170 175Thr Ser Pro Lys Arg Trp Ile Tyr Asp 180 18557115PRTArtificial sequencesynthetic sequence 57Pro Lys Lys Lys Arg Lys Val Asp Ala Leu Asp Asp Phe Asp Leu Asp1 5 10 15Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly 20 25 30Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala 35 40 45Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Gly Gly Ser Gly Gly 50 55 60Ser Gly Gly Ser Leu Glu Ile Glu Ala Ala Phe Leu Glu Arg Glu Asn65 70 75 80Thr Ala Leu Glu Thr Arg Val Ala Glu Leu Arg Gln Arg Val Gln Arg 85 90 95Leu Arg Asn Arg Val Ser Gln Tyr Arg Thr Arg Tyr Gly Pro Leu Gly 100 105 110Gly Gly Lys 11558198PRTArtificial sequencesynthetic sequence 58Pro Gly Leu Leu Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met1 5 10 15Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu 20 25 30Ala Asp Phe Asn Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg 35 40 45Met Asp Val Gly Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser 50 55 60Tyr Met Val Thr Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly65 70 75 80Asp Ile Leu Gln Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg 85 90 95Thr Asn Thr Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu 100 105 110Val Ser Gln Gly Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val 115 120 125Thr Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile 130 135 140Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu145 150 155 160Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu 165 170 175Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser 180 185 190Gly Glu Gly Leu Gln Ile 19559198PRTArtificial sequencesynthetic sequence 59Pro Gly Leu Leu Gly Val Lys Lys Leu Gly Pro Lys Arg Asn Ser Met1 5 10 15Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu 20 25 30Ala Asp Phe Asn Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg 35 40 45Met Asp Val Gly Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser 50 55 60Tyr Met Val Thr Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly65 70 75 80Asp Ile Leu Gln Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg 85 90 95Thr Asn Thr Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu 100 105 110Val Ser Gln Gly Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val 115 120 125Thr Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile 130 135 140Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu145 150 155 160Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu 165 170 175Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser 180 185 190Gly Glu Gly Leu Gln Ile 19560199PRTArtificial sequencesynthetic sequence 60Pro Gly Ile Ala Gly Ile Ser Ser Pro Gly Pro Lys Arg Lys Ser Met1 5 10 15Val Leu Asp Val Val Phe Val Leu Glu Gly Ser Asp Glu Val Gly Glu 20 25 30Ala Asn Phe Asn Lys Ser Lys Glu Phe Val Glu Glu Val Ile Gln Arg 35 40 45Met Asp Val Ser Pro Asp Ala Thr Arg Ile Ser Val Leu Gln Tyr Ser 50 55 60Tyr Thr Val Thr Met Glu Tyr Ala Phe Asn Gly Ala Gln Ser Lys Glu65 70 75 80Glu Val Leu Arg His Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg 85 90 95Thr Asn Thr Gly Gln Ala Leu Gln Tyr Leu Ser Glu His Ser Phe Ser 100 105 110Pro Ser Gln Gly Asp Arg Val Glu Ala Pro Asn Leu Val Tyr Met Val 115 120 125Thr Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile 130 135 140Gln Val Val Pro Ile Gly Val Gly Pro His Ala Asn Met Gln Glu Leu145 150 155 160Glu Arg Ile Ser Arg Pro Ile Ala Pro Ile Phe Ile Arg Asp Phe Glu 165 170 175Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Thr Cys Cys Ser 180 185 190Lys Glu Gly Leu Gln Leu Pro 19561199PRTArtificial sequencesynthetic sequence 61Pro Gly Ile Ala Gly Thr Leu Ser Pro Gly Pro Lys Arg Lys Ser Met1 5 10 15Val Leu Asp Val Val Phe Val Leu Glu Gly Ser Asp Glu Val Gly Glu 20 25 30Ala Asn Phe Asn Lys Ser Lys Glu Phe Val Glu Glu Val Ile Gln Arg 35 40 45Met Asp Val Ser Pro Asp Ala Thr Arg Ile Ser Val Leu Gln Tyr Ser 50 55 60Tyr Thr Val Thr Met Glu Tyr Ala Phe Asn Gly Ala Gln Ser Lys Glu65 70 75 80Glu Val Leu Arg His Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg 85 90 95Thr Asn Thr Gly Gln Ala Leu Gln Tyr Leu Ser Glu His Ser Phe Ser 100 105 110Pro Ser Gln Gly Asp Arg Val Glu Ala Pro Asn Leu Val Tyr Met Val 115 120 125Thr Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile 130 135 140Gln Val Val Pro Ile Gly Val Gly Pro His Ala Asn Met Gln Glu Leu145 150 155 160Glu Arg Ile Ser Arg Pro Ile Ala Pro Ile Phe Ile Arg Asp Phe Glu 165 170 175Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gln Thr Cys Cys Ser 180 185 190Lys Glu Gly Leu Gln Leu Pro 19562430PRTArtificial sequencesynthetic sequence 62Met Val Ser Lys Gly Glu Glu Asp Asn Ser Asp Val Trp Trp Gly Gly1 5 10 15Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val 20 25 30Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp 35 40 45Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu 50 55 60Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr65 70 75 80Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr 85 90 95Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly 100 105 110Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu 115 120 125Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu 130 135 140Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val145 150 155 160Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala 165 170 175Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala 180 185 190Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala 195 200 205Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys 210 215 220Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His225 230 235 240Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg 245 250 255Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln 260 265 270Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln 275 280 285Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp 290 295 300Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp305 310 315 320Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu 325 330 335Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser 340 345 350Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro 355 360 365Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly 370 375 380Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Asn Leu385 390 395 400Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 405 410 415Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Arg 420 425 43063194PRTArtificial sequencesynthetic sequence 63Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro1 5 10 15Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 20 25 30Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 35 40 45Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 50 55 60Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu65 70 75 80Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 85 90 95Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 100 105 110His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 115 120 125Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 130 135 140Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe145 150 155 160Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 165 170 175Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 180 185 190Val Arg64461PRTArtificial sequencesynthetic sequence 64Pro Arg His Ser Lys Ser His Ala Ala Ala Gln Lys Gln Asn Asn Trp1 5 10 15Ile Trp Ser Trp Phe Gly Asn Ser Gln Ser Thr Thr Gln Thr Gln Glu 20 25 30Pro Thr Thr Ser Ala Thr Thr Ala Leu Met Thr Thr Pro Glu Thr Pro 35 40 45Pro Lys Arg Gln Glu Ala Gln Asn Trp Phe Ser Trp Leu Phe Gln Pro 50 55 60Ser Glu Ser Lys Ser His Leu His Thr Thr Thr Lys Met Pro Gly Thr65 70 75 80Glu Ser Asn Thr Asn Pro Thr Gly Trp Glu Pro Asn Glu Glu Asn Glu 85 90 95Asp Glu Thr Asp Lys Tyr Pro Ser Phe Ser Gly Ser Gly Ile Asp Asp 100 105 110Asp Glu Asp Phe Ile Ser Ser Thr Ile Ala Ser Thr Pro Arg Val Ser 115 120 125Ala Arg Thr Glu Asp Asn Gln Asp Trp Thr Gln Trp Lys Pro Asn His 130 135 140Ser Asn Pro Glu Val Leu Leu

Gln Thr Thr Thr Arg Met Ala Asp Ile145 150 155 160Asp Arg Ile Ser Thr Ser Ala His Gly Glu Asn Trp Thr Pro Glu Pro 165 170 175Gln Pro Pro Phe Asn Asn His Glu Tyr Gln Asp Glu Glu Glu Thr Pro 180 185 190His Ala Thr Ser Thr Thr Pro Asn Ser Thr Ala Glu Ala Ala Ala Thr 195 200 205Gln Gln Glu Thr Trp Phe Gln Asn Gly Trp Gln Gly Lys Asn Pro Pro 210 215 220Thr Pro Ser Glu Asp Ser His Val Thr Glu Gly Thr Thr Ala Ser Ala225 230 235 240His Asn Asn His Pro Ser Gln Arg Ile Thr Thr Gln Ser Gln Glu Asp 245 250 255Val Ser Trp Thr Asp Phe Phe Asp Pro Ile Ser His Pro Met Gly Gln 260 265 270Gly His Gln Thr Glu Ser Lys Asp Thr Asp Ser Ser His Ser Thr Thr 275 280 285Leu Gln Pro Thr Ala Ala Pro Asn Thr His Leu Val Glu Asp Leu Asn 290 295 300Arg Thr Gly Pro Leu Ser Val Thr Thr Pro Gln Ser His Ser Gln Asn305 310 315 320Phe Ser Thr Leu His Gly Glu Pro Glu Glu Asp Glu Asn His Pro Thr 325 330 335Thr Ser Ile Leu Pro Ser Ser Thr Lys Ser Gly Ala Lys Asp Ala Arg 340 345 350Arg Gly Gly Ser Leu Pro Thr Asp Thr Thr Thr Ser Val Glu Gly Tyr 355 360 365Thr Phe Gln Tyr Pro Asp Thr Met Glu Asn Gly Thr Leu Phe Pro Val 370 375 380Thr Pro Ala Lys Thr Glu Val Phe Gly Glu Thr Glu Val Thr Leu Ala385 390 395 400Thr Asp Ser Asn Val Asn Val Asp Gly Ser Leu Pro Gly Asp Arg Asp 405 410 415Ser Ser Lys Asp Ser Arg Gly Ser Ser Arg Thr Val Thr His Gly Ser 420 425 430Glu Leu Ala Gly His Ser Ser Ala Asn Gln Asp Ser Gly Val Thr Thr 435 440 445Thr Ser Gly Pro Met Arg Arg Pro Gln Ile Pro Glu Arg 450 455 4606534PRTArtificial sequencesynthetic sequence 65Pro Arg Asp Glu Glu Ser Tyr Asp Ser Val Thr Phe Asp Ala His Gln1 5 10 15Tyr Gly Ala Thr Thr Gln Ala Arg Ala Asp Gly Leu Ala Asn Ala Gln 20 25 30Val Arg66163PRTArtificial sequencesynthetic sequence 66Ala Glu Met Asp Arg Glu Asp Ala Glu His Val Lys Asn Ser Thr Tyr1 5 10 15Val Ala Leu Ile Ile Ala Thr Asp Asp Gly Ser Pro Ile Ala Thr Gly 20 25 30Thr Gly Thr Leu Leu Leu Val Leu Leu Asp Val Asn Asp Asn Ala Pro 35 40 45Ile Pro Glu Pro Arg Asn Met Gln Phe Cys Gln Arg Asn Pro Gln Pro 50 55 60His Ile Ile Thr Ile Leu Asp Pro Asp Leu Pro Pro Asn Thr Ser Pro65 70 75 80Phe Thr Ala Glu Leu Thr His Gly Ala Ser Val Asn Trp Thr Ile Glu 85 90 95Tyr Asn Asp Ala Ala Gln Glu Ser Leu Ile Leu Gln Pro Arg Lys Asp 100 105 110Leu Glu Ile Gly Glu Tyr Lys Ile His Leu Lys Leu Ala Asp Asn Gln 115 120 125Asn Lys Asp Gln Val Thr Thr Leu Asp Val His Val Cys Asp Cys Glu 130 135 140Gly Thr Val Asn Asn Cys Met Lys Ala Gly Ile Val Ala Ala Gly Leu145 150 155 160Gln Val Arg67342PRTArtificial sequencesynthetic sequence 67Asn Gly Ala Ile Phe Gln Glu Thr Leu Ser Gly Ala Glu Ser Thr Ser1 5 10 15Leu Val Ala Ala Arg Gly Ser Cys Ile Ala Asn Ala Glu Glu Val Asp 20 25 30Val Pro Ile Lys Leu Tyr Cys Asn Gly Asp Gly Glu Trp Leu Val Pro 35 40 45Ile Gly Arg Cys Met Cys Lys Ala Gly Phe Glu Ala Val Glu Asn Gly 50 55 60Thr Val Cys Arg Gly Cys Pro Ser Gly Thr Phe Lys Ala Asn Gln Gly65 70 75 80Asp Glu Ala Cys Thr His Cys Pro Ile Asn Ser Arg Thr Thr Ser Glu 85 90 95Gly Ala Thr Asn Cys Val Cys Arg Asn Gly Tyr Tyr Arg Ala Asp Leu 100 105 110Asp Pro Leu Asp Met Pro Cys Thr Thr Ile Pro Ser Ala Pro Gln Ala 115 120 125Val Ile Ser Ser Val Asn Glu Thr Ser Leu Met Leu Glu Trp Thr Pro 130 135 140Pro Arg Asp Ser Gly Gly Arg Glu Asp Leu Val Tyr Asn Ile Ile Cys145 150 155 160Lys Ser Cys Gly Ser Gly Arg Gly Ala Cys Thr Arg Cys Gly Asp Asn 165 170 175Val Gln Tyr Ala Pro Arg Gln Leu Gly Leu Thr Glu Pro Arg Ile Tyr 180 185 190Ile Ser Asp Leu Leu Ala His Thr Gln Tyr Thr Phe Glu Ile Gln Ala 195 200 205Val Asn Gly Val Thr Asp Gln Ser Pro Phe Ser Pro Gln Phe Ala Ser 210 215 220Val Asn Ile Thr Thr Asn Gln Ala Ala Pro Ser Ala Val Ser Ile Met225 230 235 240His Gln Val Ser Arg Thr Val Asp Ser Ile Thr Leu Ser Trp Ser Gln 245 250 255Pro Asp Gln Pro Asn Gly Val Ile Leu Asp Tyr Glu Leu Gln Tyr Tyr 260 265 270Glu Lys Gln Glu Leu Ser Glu Tyr Asn Ala Thr Ala Ile Lys Ser Pro 275 280 285Thr Asn Thr Val Thr Val Gln Gly Leu Lys Ala Gly Ala Ile Tyr Val 290 295 300Phe Gln Val Arg Ala Arg Thr Val Ala Gly Tyr Gly Arg Tyr Ser Gly305 310 315 320Lys Met Tyr Phe Gln Thr Met Thr Glu Ala Glu Tyr Gln Thr Ser Ile 325 330 335Lys Glu Lys Leu Pro Arg 34068110PRTArtificial sequencesynthetic sequence 68Ala Pro Ser Ala Val Ser Ile Met His Gln Val Ser Arg Thr Val Asp1 5 10 15Ser Ile Thr Leu Ser Trp Ser Gln Pro Asp Gln Pro Asn Gly Val Ile 20 25 30Leu Asp Tyr Glu Leu Gln Tyr Tyr Glu Lys Gln Glu Leu Ser Glu Tyr 35 40 45Asn Ala Thr Ala Ile Lys Ser Pro Thr Asn Thr Val Thr Val Gln Gly 50 55 60Leu Lys Ala Gly Ala Ile Tyr Val Phe Gln Val Arg Ala Arg Thr Val65 70 75 80Ala Gly Tyr Gly Arg Tyr Ser Gly Lys Met Tyr Phe Gln Thr Met Thr 85 90 95Glu Ala Glu Tyr Gln Thr Ser Ile Lys Glu Lys Leu Pro Arg 100 105 11069233PRTArtificial sequencesynthetic sequence 69Met Ala Met Ala Arg Ser Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp1 5 10 15Gly Leu Leu Met Val Leu Cys Arg Thr Ala Ile Ser Arg Ser Ile Val 20 25 30Leu Glu Pro Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly 35 40 45Gln Gly Leu Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile 50 55 60Cys Pro Lys Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys65 70 75 80Val Tyr Met Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys 85 90 95Glu Asn Thr Pro Leu Leu Asn Cys Ala Arg Pro Asp Gln Asp Val Lys 100 105 110Phe Thr Ile Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu 115 120 125Phe Gln Lys Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser 130 135 140Leu Glu Gly Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala145 150 155 160Met Lys Ile Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser 165 170 175Ala Arg Asn His Gly Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr 180 185 190Asn Gly Arg Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly 195 200 205Ser Ser Thr Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Leu Leu 210 215 220Gly Ser Glu Val Ala Leu Phe Ala Arg225 2307047PRTArtificial sequencesynthetic sequence 70Val Tyr Val Arg Pro Thr Asn Glu Thr Leu Tyr Glu Ala Pro Glu Pro1 5 10 15Ile Phe Thr Ser Asn Ser Ser Cys Ser Gly Leu Gly Gly Cys His Leu 20 25 30Phe Leu Thr Thr Val Pro Val Leu Trp Ser Leu Leu Gly Ser Arg 35 40 457166PRTArtificial sequencesynthetic sequence 71Gly Gln Asp Ala Ser Ser Ala Gly Ser Ala Arg Asn His Gly Pro Thr1 5 10 15Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg Ser Ser Thr Thr 20 25 30Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr Asp Gly Asn Ser 35 40 45Ala Gly His Ser Gly Asn Asn Leu Leu Gly Ser Glu Val Ala Leu Phe 50 55 60Ala Arg6572211PRTArtificial sequencesynthetic sequence 72Asp Asn Gln Gln Met Arg Glu Arg Arg Ala Val Ser Asn Phe Ser Thr1 5 10 15Ala Ser Gln Ile Tyr Glu Ala Pro Lys Met Leu Ser Met Leu Phe Arg 20 25 30Thr Tyr Lys Asp Gln Gly Gln Ile Leu Tyr Ala Ala Thr Asn Gln Met 35 40 45Phe Thr Ser Leu Ser Leu Arg Glu Gly Arg Leu Val Tyr Tyr Ser Lys 50 55 60Gln His Leu Thr Ile Asn Met Thr Val Gln Glu Thr Ser Thr Leu Asn65 70 75 80Asp Gly Lys Trp His Asn Val Ser Leu Phe Ser Glu Ser Arg Ser Leu 85 90 95Arg Leu Ile Val Asp Gly Arg Gln Val Gly Asp Glu Leu Asp Ile Ala 100 105 110Gly Val His Asp Phe Leu Asp Pro Tyr Leu Thr Ile Leu Asn Val Gly 115 120 125Gly Glu Ala Phe Val Gly Cys Leu Ala Asn Val Thr Val Asn Asn Glu 130 135 140Leu Gln Pro Leu Asn Gly Ser Gly Ser Ile Phe Pro Glu Val Arg Tyr145 150 155 160His Gly Lys Ile Glu Ser Gly Cys Arg Gly Asp Ile Gly Gln Asp Ala 165 170 175Ala Gln Val Ala Asp Pro Leu Ser Ile Gly Phe Thr Leu Val Ile Val 180 185 190Phe Phe Val Ile Leu Val Val Ala Ile Leu Gly Ser Tyr Val Ile Tyr 195 200 205Arg Phe Arg 21073370PRTArtificial sequencesynthetic sequence 73Met Ala Cys Lys Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr1 5 10 15Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala 20 25 30Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile 35 40 45Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn 50 55 60Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70 75 80Glu Leu Gly Gly Ser Gly Gly Ser Gly Glu Gly Arg Val Lys Glu Ser 85 90 95Ile Thr Arg Arg Arg Arg Ala Pro Ser Val Ala Asn Val Gly Ser His 100 105 110Ser Asp Leu Ser Leu Lys Ile Pro Glu Ile Ser Ile Gln Asp Met Thr 115 120 125Ala Gln Val Thr Ser Pro Ser Gly Lys Thr His Glu Ala Glu Ile Val 130 135 140Glu Gly Glu Asn His Thr Tyr Ser Ile Arg Phe Val Pro Ala Glu Met145 150 155 160Gly Thr His Thr Val Ser Val Lys Tyr Lys Gly Gln His Val Pro Gly 165 170 175Ser Pro Phe Gln Phe Thr Val Gly Pro Leu Gly Glu Gly Gly Ala His 180 185 190Lys Val Arg Ala Gly Gly Pro Gly Leu Glu Arg Ala Glu Ala Gly Val 195 200 205Pro Ala Glu Phe Ser Ile Trp Thr Arg Glu Ala Gly Ala Gly Gly Leu 210 215 220Ala Ile Ala Val Glu Gly Pro Ser Lys Ala Glu Ile Ser Phe Glu Asp225 230 235 240Arg Lys Asp Gly Ser Ser Gly Val Ala Tyr Val Val Gln Glu Pro Gly 245 250 255Asp Tyr Glu Val Ser Val Lys Phe Asn Glu Glu His Ile Pro Asp Ser 260 265 270Pro Phe Val Val Pro Val Ala Ser Pro Ser Ser Gly Gly Ser Gly Gly 275 280 285Thr Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu 290 295 300Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln305 310 315 320Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 325 330 335Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys 340 345 350Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Lys Cys Leu 355 360 365Glu Arg 37074293PRTArtificial sequencesynthetic sequence 74Met Ala Cys Lys Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr1 5 10 15Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala 20 25 30Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile 35 40 45Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn 50 55 60Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70 75 80Glu Leu Gly Gly Ser Gly Gly Pro Thr Phe Arg Ser Ser Leu Phe Leu 85 90 95Trp Val Arg Pro Gly Gly Ser Gly Gly Ser Gly Pro Leu Gly Glu Gly 100 105 110Gly Ala His Lys Val Arg Ala Gly Gly Pro Gly Leu Glu Arg Ala Glu 115 120 125Ala Gly Val Pro Ala Glu Phe Ser Ile Trp Thr Arg Glu Ala Gly Ala 130 135 140Gly Gly Leu Ala Ile Ala Val Glu Gly Pro Ser Lys Ala Glu Ile Ser145 150 155 160Phe Glu Asp Arg Lys Asp Gly Ser Cys Gly Val Ala Tyr Val Val Gln 165 170 175Glu Pro Gly Asp Tyr Glu Val Ser Val Lys Phe Asn Glu Glu His Ile 180 185 190Pro Asp Ser Pro Phe Val Val Pro Val Ala Ser Pro Ser Ser Gly Gly 195 200 205Ser Gly Gly Thr Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr 210 215 220Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala225 230 235 240Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile 245 250 255Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn 260 265 270Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 275 280 285Lys Cys Leu Glu Arg 29075515PRTArtificial sequencesynthetic sequence 75Met Val Ser Lys Gly Glu Glu Thr Thr Met Gly Val Ile Lys Pro Asp1 5 10 15Met Lys Ile Lys Leu Lys Met Glu Gly Asn Val Asn Gly His Ala Phe 20 25 30Val Ile Glu Gly Glu Gly Glu Gly Lys Pro Tyr Asp Gly Thr Asn Thr 35 40 45Ile Asn Leu Glu Val Lys Glu Gly Ala Pro Leu Pro Phe Ser Tyr Asp 50 55 60Ile Leu Thr Thr Ala Phe Ala Tyr Gly Asn Arg Ala Phe Thr Lys Tyr65 70 75 80Pro Asp Asp Ile Pro Asn Tyr Phe Lys Gln Ser Phe Pro Glu Gly Tyr 85 90 95Ser Trp Glu Arg Thr Met Thr Phe Glu Asp Lys Gly Ile Val Lys Val 100 105 110Lys Ser Asp Ile Ser Met Glu Glu Asp Ser Phe Ile Tyr Glu Ile His 115 120 125Leu Lys Gly Glu Asn Phe Pro Pro Asn Gly Pro Val Met Gln Lys Lys 130 135 140Thr Thr Gly Trp Asp Ala Ser Thr Glu Arg Met Tyr Val Arg Asp Gly145 150 155 160Val Leu Lys Gly Asp Val Lys His Lys Leu Leu Leu Glu Gly Gly Gly 165 170 175His His Arg Val Asp Phe Lys Thr Ile Tyr Arg Ala Lys Lys Ala Val 180 185 190Lys Leu Pro Asp Tyr His Phe Val Asp His Arg Ile Glu Ile Leu Asn 195 200 205His Asp Lys Asp Tyr Asn Lys Val Thr Val Tyr Glu Ser Ala Val Ala 210 215 220Arg Asn Ser Thr Asp Gly Met Asp Glu Leu Tyr

Lys Gly Pro Gly Gly225 230 235 240Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala 245 250 255Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 260 265 270Pro Gly Gly Ala Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 275 280 285Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 290 295 300Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr305 310 315 320Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 325 330 335Leu Val Thr Thr Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro 340 345 350Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 355 360 365Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 370 375 380Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile385 390 395 400Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 405 410 415Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp 420 425 430Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile 435 440 445Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 450 455 460Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr465 470 475 480Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 485 490 495Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 500 505 510Leu Tyr Lys 51576328PRTArtificial sequencesynthetic sequence 76Ala Ala Arg Ser Cys Arg Phe Arg Gly Arg His Tyr Lys Arg Glu Phe1 5 10 15Arg Leu Glu Gly Glu Pro Val Ala Leu Arg Cys Pro Gln Val Pro Tyr 20 25 30Trp Leu Trp Ala Ser Val Ser Pro Arg Ile Asn Leu Thr Trp His Lys 35 40 45Asn Asp Ser Ala Arg Thr Val Pro Gly Glu Glu Glu Thr Arg Met Trp 50 55 60Ala Gln Asp Gly Ala Leu Trp Leu Leu Pro Ala Leu Gln Glu Asp Ser65 70 75 80Gly Thr Tyr Val Cys Thr Thr Arg Asn Ala Ser Tyr Cys Asp Lys Met 85 90 95Ser Ile Glu Leu Arg Val Phe Glu Asn Thr Asp Ala Phe Leu Pro Phe 100 105 110Ile Ser Tyr Pro Gln Ile Leu Thr Leu Ser Thr Ser Gly Val Leu Val 115 120 125Cys Pro Asp Leu Ser Glu Phe Thr Arg Asp Lys Thr Asp Val Lys Ile 130 135 140Gln Trp Tyr Lys Asp Ser Leu Leu Leu Asp Lys Asp Asn Glu Lys Phe145 150 155 160Leu Ser Val Arg Gly Thr Thr His Leu Leu Val His Asp Val Ala Leu 165 170 175Glu Asp Ala Gly Tyr Tyr Arg Cys Val Leu Thr Phe Ala His Glu Gly 180 185 190Gln Gln Tyr Asn Ile Thr Arg Ser Ile Glu Leu Arg Ile Lys Lys Lys 195 200 205Lys Glu Glu Thr Ile Pro Val Ile Ile Ser Pro Leu Lys Thr Ile Ser 210 215 220Ala Ser Leu Gly Ser Arg Leu Thr Ile Pro Cys Lys Val Phe Leu Gly225 230 235 240Thr Gly Thr Pro Leu Thr Thr Met Leu Trp Trp Thr Ala Asn Asp Thr 245 250 255His Ile Glu Ser Ala Tyr Pro Gly Gly Arg Val Thr Glu Gly Pro Arg 260 265 270Gln Glu Tyr Ser Glu Asn Asn Glu Asn Tyr Ile Glu Val Pro Leu Ile 275 280 285Phe Asp Pro Val Thr Arg Glu Asp Leu His Met Asp Phe Lys Cys Val 290 295 300Val His Asn Thr Leu Ser Phe Gln Thr Leu Arg Thr Thr Val Lys Glu305 310 315 320Ala Ser Ser Thr Phe Ser Gly Arg 32577232PRTArtificial sequencesynthetic sequence 77Lys Arg Pro Lys Pro Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro1 5 10 15Gly Gln Gly Ser Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Thr 20 25 30Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Ser 35 40 45Trp Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Gly 50 55 60Trp Gly Gln Gly Gly Gly Thr His Asn Gln Trp Asn Lys Pro Ser Lys65 70 75 80Pro Lys Thr Asn Leu Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala 85 90 95Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg 100 105 110Pro Met Ile His Phe Gly Asn Asp Trp Glu Asp Arg Tyr Tyr Arg Glu 115 120 125Asn Met Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln 130 135 140Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Ile Thr Ile145 150 155 160Lys Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu 165 170 175Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys Val Thr 180 185 190Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg Ser Ser 195 200 205Ser Thr Val Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser Phe 210 215 220Leu Ile Phe Leu Ile Val Gly Arg225 2307841PRTArtificial sequencesynthetic sequence 78Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys His Leu1 5 10 15Gly Ile Glu Phe Met Glu Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu 20 25 30Thr Ile Thr Gly Ile Cys Ile Ala Arg 35 407973PRTArtificial sequencesynthetic sequence 79Pro Arg Asn Pro Gln Cys Val Arg Trp Asn Ser Phe Thr Asn Arg Trp1 5 10 15Thr Arg Leu Gly Cys Gln Thr Glu Ile Pro Asp Phe Asp Gly Asp Phe 20 25 30Asn Pro Ala Ala Gln Gln Ala Ile Leu Val Asn Cys Ser Cys Thr His 35 40 45Ile Ser Ser Tyr Ala Val Ile Val Asp Val Ile Asp Pro Glu Asp Ile 50 55 60Pro Glu Pro Ser Leu Leu Val Gln Arg65 7080116PRTArtificial sequencesynthetic sequence 80Ile Thr Tyr Pro Ser Glu Gln Met Gln Gln Ser Glu Gln Val Val Tyr1 5 10 15Arg Ser Leu Gly Ser Pro His Leu Ala Gln Pro Ile Lys Leu Gln Met 20 25 30Trp Leu Asp Val Asp Ser Ala Arg Phe Gly Pro Arg Ser Asn Pro Gln 35 40 45Cys Val Arg Trp Asn Ser Phe Thr Asn Arg Trp Thr Arg Leu Gly Cys 50 55 60Gln Thr Glu Ile Pro Asp Phe Asp Gly Asp Phe Asn Pro Ala Ala Gln65 70 75 80Gln Ala Ile Leu Val Asn Cys Ser Cys Thr His Ile Ser Ser Tyr Ala 85 90 95Val Ile Val Asp Val Ile Asp Pro Glu Asp Ile Pro Glu Pro Ser Leu 100 105 110Leu Val Gln Arg 1158144PRTArtificial sequencesynthetic sequence 81Pro Arg Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly1 5 10 15Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala 20 25 30Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Arg Arg 35 40828PRTArtificial sequencesynthetic sequence 82Pro Pro Ala Asn Val Lys Tyr Val1 58321PRTArtificial sequencesynthetic sequence 83Phe Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly1 5 10 15Cys Gly Val Leu Leu 208421PRTArtificial sequencesynthetic sequence 84Leu Leu Tyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Ile Ile1 5 10 15Leu Leu Gly Val Ile 208521PRTArtificial sequencesynthetic sequence 85Leu Pro Leu Leu Val Ala Gly Ala Val Leu Leu Leu Val Ile Leu Val1 5 10 15Leu Gly Val Met Val 208621PRTArtificial sequencesynthetic sequence 86Pro Val Leu Cys Ser Pro Val Ala Gly Val Ile Leu Leu Ala Leu Gly1 5 10 15Ala Leu Leu Val Leu 208721PRTArtificial sequencesynthetic sequence 87Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly1 5 10 15Cys Gly Val Leu Leu 208821PRTArtificial sequencesynthetic sequence 88Leu Leu Tyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Phe Ile1 5 10 15Leu Leu Gly Val Ile 208921PRTArtificial sequencesynthetic sequence 89Leu Leu Pro Leu Leu Val Ala Gly Ala Val Phe Leu Leu Ile Ile Phe1 5 10 15Ile Leu Gly Val Met 209021PRTArtificial sequencesynthetic sequence 90Pro Ile Leu Cys Ser Pro Val Val Gly Val Leu Leu Leu Ala Leu Gly1 5 10 15Ala Leu Leu Val Leu 209123PRTArtificial sequencesynthetic sequence 91Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe1 5 10 15Val Gly Cys Gly Val Leu Leu 209221PRTArtificial sequencesynthetic sequence 92Leu Leu Tyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Leu Ile1 5 10 15Leu Leu Gly Val Ile 209321PRTArtificial sequencesynthetic sequence 93Leu Pro Leu Leu Val Ala Gly Ala Val Phe Leu Leu Val Ile Phe Val1 5 10 15Leu Gly Val Met Val 20948PRTArtificial sequencesynthetic sequence 94Val Gly Cys Gly Val Leu Leu Ser1 5957PRTArtificial sequencesynthetic sequence 95Gly Cys Gly Val Leu Leu Ser1 596333PRTArtificial sequencesynthetic sequence 96Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro1 5 10 15Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala 20 25 30Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp 35 40 45Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys 50 55 60Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp65 70 75 80Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln 85 90 95Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp 100 105 110His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys 115 120 125Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala 130 135 140Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg145 150 155 160Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr 165 170 175Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro 180 185 190Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser 195 200 205Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly 210 215 220Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val225 230 235 240Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys 245 250 255Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser 260 265 270Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu 275 280 285Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 290 295 300Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu305 310 315 320Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu 325 33097370PRTArtificial sequencesynthetic sequence 97Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro1 5 10 15Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn 20 25 30Gly Leu Leu Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly 35 40 45Arg Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu 50 55 60Cys Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn65 70 75 80His Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp 85 90 95Pro Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser 100 105 110Asp Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp 115 120 125Gly Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp 130 135 140Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys145 150 155 160Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val 165 170 175Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro 180 185 190Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser 195 200 205His Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln 210 215 220Gln Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His225 230 235 240Pro Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro 245 250 255Gly Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile 260 265 270Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln 275 280 285Ser Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu 290 295 300Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu305 310 315 320Ala Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His 325 330 335Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 340 345 350Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln 355 360 365Lys Leu 37098787PRTArtificial sequencesynthetic sequence 98Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1 5 10 15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70 75 80Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145 150 155 160Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Ile Leu 195 200 205Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro Gln 210 215 220Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly Asn225 230 235 240Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp Gly 245 250

255Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr Gln 260 265 270Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser Gln 275 280 285Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr 290 295 300Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe305 310 315 320Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp 325 330 335Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly 340 345 350Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn 355 360 365Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn Val 370 375 380Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr385 390 395 400Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val 405 410 415Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln 420 425 430Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu 435 440 445Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe Gln 450 455 460Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly465 470 475 480Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val 485 490 495Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala 500 505 510Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg 515 520 525Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser Arg Leu Asp 530 535 540Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val Gly545 550 555 560Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val Glu 565 570 575Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu Asp 580 585 590Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys Pro 595 600 605Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys Ser 610 615 620Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His Leu625 630 635 640Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln Leu 645 650 655Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr Ala 660 665 670Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp Gln 675 680 685Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp Ser 690 695 700Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln Gly705 710 715 720Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly Leu 725 730 735Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu Asp 740 745 750Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp 755 760 765Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met 770 775 780Leu Pro Gly78599889PRTArtificial sequencesynthetic sequence 99Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Ala 20 25 30Gln Pro Ala Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly 35 40 45Asp Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr 50 55 60Gln Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys65 70 75 80Leu Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala 85 90 95Ile Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr 100 105 110Leu Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp 115 120 125Thr Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser 130 135 140Asp Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly145 150 155 160Pro Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp 165 170 175Ala Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro 180 185 190Pro Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala 195 200 205Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val 210 215 220Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys225 230 235 240Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met 245 250 255Trp Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp 260 265 270Ala Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His 275 280 285Leu Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr 290 295 300Gly Gly Trp Lys Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg305 310 315 320Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys 325 330 335Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His 340 345 350Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro 355 360 365Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp 370 375 380Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly385 390 395 400Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln 405 410 415Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn 420 425 430Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro 435 440 445Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro 450 455 460Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His465 470 475 480Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln 485 490 495Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro 500 505 510Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly 515 520 525Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg 530 535 540Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser545 550 555 560Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly 565 570 575Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala 580 585 590Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 595 600 605Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys 610 615 620Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys625 630 635 640Leu Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu 645 650 655Leu Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala 660 665 670Gln Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn 675 680 685Lys Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His 690 695 700His Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu705 710 715 720Arg Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp 725 730 735Gly Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu 740 745 750Thr Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu 755 760 765Glu Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly 770 775 780Cys Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu785 790 795 800Thr Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu 805 810 815Leu Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu 820 825 830Leu Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly 835 840 845Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp 850 855 860Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp865 870 875 880Asp Phe Asp Leu Asp Met Leu Pro Gly 885100854PRTArtificial sequencesynthetic sequence 100Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr65 70 75 80His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser145 150 155 160Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180 185 190Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr225 230 235 240Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser 595 600 605Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn 610 615 620Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu625 630 635 640Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala 645 650 655Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His 660 665 670Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn 675 680 685Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys 690 695 700Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu705 710 715 720Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala 725 730 735Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu 740 745 750Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr 755 760 765Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp 770 775 780His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile785 790 795 800Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp 805 810 815Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp 820 825 830Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp 835 840 845Leu Asp Met Leu Pro Gly 850101854PRTArtificial sequencesynthetic sequence 101Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr65 70 75 80His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser145 150 155 160Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180

185 190Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr225 230 235 240Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser 595 600 605Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn 610 615 620Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu625 630 635 640Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala 645 650 655Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His 660 665 670Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn 675 680 685Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys 690 695 700Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu705 710 715 720Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala 725 730 735Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu 740 745 750Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr 755 760 765Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp 770 775 780His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile785 790 795 800Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp 805 810 815Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp 820 825 830Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp 835 840 845Leu Asp Met Leu Pro Gly 850102854PRTArtificial sequencesynthetic sequence 102Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gln 20 25 30Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser 35 40 45Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr 50 55 60Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly65 70 75 80Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg 85 90 95Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 100 105 110Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 115 120 125Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr 130 135 140Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met 165 170 175Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser 180 185 190Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro 195 200 205Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly 210 215 220Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser225 230 235 240Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 245 250 255Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys Ala 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser 595 600 605Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn 610 615 620Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu625 630 635 640Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala 645 650 655Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His 660 665 670Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn 675 680 685Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys 690 695 700Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu705 710 715 720Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala 725 730 735Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu 740 745 750Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr 755 760 765Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp 770 775 780His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile785 790 795 800Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp 805 810 815Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp 820 825 830Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp 835 840 845Leu Asp Met Leu Pro Gly 850103871PRTArtificial sequencesynthetic sequence 103Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gln 20 25 30Val Gln Leu Gln Gln Gln Val Gln Leu Gln Glu Ser Gly Gly Asp Leu 35 40 45Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 50 55 60Thr Phe Ser His Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys65 70 75 80Arg Leu Glu Trp Val Ala Thr Ile Gly Ser Arg Gly Thr Tyr Thr His 85 90 95Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp 100 105 110Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr 115 120 125Ala Met Tyr Tyr Cys Ala Arg Arg Ser Glu Phe Tyr Tyr Tyr Gly Asn 130 135 140Thr Tyr Tyr Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly Ala Ser Val145 150 155 160Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 165 170 175Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Phe Leu Ala Val 180 185 190Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val 195 200 205Asp Asn Tyr Gly Phe Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly 210 215 220Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ile Ser Asn Arg Gly Ser Gly225 230 235 240Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu 245 250 255Asn Ile His Pro Val Glu Glu Asp Asp Pro Ala Met Tyr Phe Cys Gln 260 265 270Gln Thr Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 275 280 285Ile Lys Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile 290 295 300Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val305 310 315 320Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys 325 330 335Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys 340 345 350Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His 355 360 365Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp 370 375 380Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys385 390 395 400Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala 405 410 415Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg 420 425 430Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln 435 440 445Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu 450 455 460His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile465 470 475 480Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys 485 490 495Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser 500 505 510Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser 515 520 525Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser 530 535 540Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu545 550 555 560Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys 565 570 575Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 580 585 590Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val 595 600 605Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met 610 615 620Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu625 630 635 640Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys 645 650 655Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg 660 665 670Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr 675 680 685His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn 690 695 700Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala705 710 715 720Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu 725 730 735Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn 740 745 750Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val 755 760 765Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro 770 775 780Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe785 790 795 800Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile 805 810 815Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala 820 825 830Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu 835 840 845Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe 850 855 860Asp Leu Asp Met Leu Pro Gly865 870104873PRTArtificial sequencesynthetic sequence 104Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly 20 25 30Ser Gln Val Gln Leu Gln Gln Gln Val Gln Leu Gln Glu Ser Gly Gly 35 40 45Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser 50 55 60Gly Phe Thr Phe Ser His Tyr Gly Met Ser Trp Val Arg Gln Thr Pro65

70 75 80Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Gly Ser Arg Gly Thr Tyr 85 90 95Thr His Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 100 105 110Asn Asp Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu 115 120 125Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Ser Glu Phe Tyr Tyr Tyr 130 135 140Gly Asn Thr Tyr Tyr Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly Ala145 150 155 160Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 165 170 175Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Phe Leu 180 185 190Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu 195 200 205Ser Val Asp Asn Tyr Gly Phe Ser Phe Met Asn Trp Phe Gln Gln Lys 210 215 220Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ile Ser Asn Arg Gly225 230 235 240Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 245 250 255Ser Leu Asn Ile His Pro Val Glu Glu Asp Asp Pro Ala Met Tyr Phe 260 265 270Cys Gln Gln Thr Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys 275 280 285Leu Glu Ile Lys Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg 290 295 300Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys305 310 315 320Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His 325 330 335Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro 340 345 350Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp 355 360 365Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly 370 375 380Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln385 390 395 400Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn 405 410 415Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro 420 425 430Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro 435 440 445Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His 450 455 460Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln465 470 475 480Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro 485 490 495Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly 500 505 510Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg 515 520 525Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser 530 535 540Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly545 550 555 560Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala 565 570 575Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 580 585 590Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys 595 600 605Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys 610 615 620Leu Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu625 630 635 640Leu Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala 645 650 655Gln Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn 660 665 670Lys Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His 675 680 685His Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu 690 695 700Arg Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp705 710 715 720Gly Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu 725 730 735Thr Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu 740 745 750Glu Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly 755 760 765Cys Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu 770 775 780Thr Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu785 790 795 800Leu Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu 805 810 815Leu Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly 820 825 830Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp 835 840 845Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp 850 855 860Asp Phe Asp Leu Asp Met Leu Pro Gly865 870105739PRTArtificial sequencesynthetic sequence 105Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 20 25 30Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 35 40 45Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr 50 55 60Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe65 70 75 80Val Ala Arg Ile Thr Trp Ser Ala Gly Tyr Thr Ala Tyr Ser Asp Ser 85 90 95Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Val 100 105 110Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 115 120 125Cys Ala Ser Arg Ser Ala Gly Tyr Ser Ser Ser Leu Thr Arg Arg Glu 130 135 140Asp Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile Leu145 150 155 160Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro Gln 165 170 175Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly Asn 180 185 190Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp Gly 195 200 205Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr Gln 210 215 220Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser Gln225 230 235 240Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr 245 250 255Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe 260 265 270Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp 275 280 285Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly 290 295 300Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn305 310 315 320Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn Val 325 330 335Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr 340 345 350Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val 355 360 365Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln 370 375 380Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu385 390 395 400Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe Gln 405 410 415Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly 420 425 430Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val 435 440 445Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala 450 455 460Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg465 470 475 480Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser Arg Leu Asp 485 490 495Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val Gly 500 505 510Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val Glu 515 520 525Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu Asp 530 535 540Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys Pro545 550 555 560Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys Ser 565 570 575Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His Leu 580 585 590Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln Leu 595 600 605Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr Ala 610 615 620Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp Gln625 630 635 640Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp Ser 645 650 655Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln Gly 660 665 670Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly Leu 675 680 685Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu Asp 690 695 700Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp705 710 715 720Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met 725 730 735Leu Pro Gly106740PRTArtificial sequencesynthetic sequence 106Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 20 25 30Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 35 40 45Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ile Ser Met 50 55 60Ala Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe65 70 75 80Val Ala Gly Ile Ser Arg Ser Ala Gly Ser Ala Val His Ala Asp Ser 85 90 95Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu 100 105 110Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr 115 120 125Cys Ala Val Arg Thr Ser Gly Phe Phe Gly Ser Ile Pro Arg Thr Gly 130 135 140Thr Ala Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile145 150 155 160Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro 165 170 175Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly 180 185 190Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp 195 200 205Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr 210 215 220Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser225 230 235 240Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu 245 250 255Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His 260 265 270Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu 275 280 285Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala 290 295 300Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn305 310 315 320Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn 325 330 335Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr 340 345 350Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr 355 360 365Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg 370 375 380Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr385 390 395 400Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe 405 410 415Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu 420 425 430Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro 435 440 445Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala 450 455 460Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser465 470 475 480Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser Arg Leu 485 490 495Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val 500 505 510Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val 515 520 525Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu 530 535 540Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys545 550 555 560Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys 565 570 575Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His 580 585 590Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln 595 600 605Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr 610 615 620Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp625 630 635 640Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp 645 650 655Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln 660 665 670Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly 675 680 685Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu 690 695 700Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe705 710 715 720Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp 725 730 735Met Leu Pro Gly 740107740PRTArtificial sequencesynthetic sequence 107Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 20 25 30Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly 35 40 45Gly Ser Leu Lys Leu Ser Cys Thr Ala Ser Val Arg Thr Leu Ser Tyr 50 55 60Tyr His Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe65 70 75 80Val Ala Gly Ile His Arg Ser Gly Glu Ser Thr Phe Tyr Ala Asp Ser 85 90 95Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 100 105 110His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 115 120 125Cys Ala Gln Arg Val Arg Gly Phe Phe Gly Pro Leu Arg Ser Thr Pro 130 135 140Ser Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile145 150 155 160Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro 165 170

175Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly 180 185 190Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp 195 200 205Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr 210 215 220Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser225 230 235 240Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu 245 250 255Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His 260 265 270Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu 275 280 285Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala 290 295 300Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn305 310 315 320Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn 325 330 335Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr 340 345 350Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr 355 360 365Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg 370 375 380Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr385 390 395 400Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe 405 410 415Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu 420 425 430Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro 435 440 445Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala 450 455 460Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser465 470 475 480Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser Arg Leu 485 490 495Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val 500 505 510Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val 515 520 525Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu 530 535 540Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys545 550 555 560Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys 565 570 575Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His 580 585 590Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln 595 600 605Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr 610 615 620Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp625 630 635 640Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp 645 650 655Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln 660 665 670Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly 675 680 685Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu 690 695 700Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe705 710 715 720Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp 725 730 735Met Leu Pro Gly 740108882PRTArtificial sequencesynthetic sequence 108Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met 20 25 30Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala Gly 35 40 45Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr 50 55 60Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Phe65 70 75 80Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp Ser 85 90 95Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg Ala Lys Asn Thr Val 100 105 110Asp Leu Gln Met Asp Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr 115 120 125Cys Ser Ala Arg Ser Arg Gly Tyr Val Leu Ser Val Leu Arg Ser Val 130 135 140Asp Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Gly145 150 155 160Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala 165 170 175Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 180 185 190Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 195 200 205Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 210 215 220Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val225 230 235 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr 245 250 255Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 260 265 270Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg Val Asn 275 280 285Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ile Leu Asp 290 295 300Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro Gln Ile305 310 315 320Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly Asn Lys 325 330 335Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp Gly Gly 340 345 350Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr Gln Ser 355 360 365Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser Gln Cys 370 375 380Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr Glu385 390 395 400Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe Ser 405 410 415Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp Asp 420 425 430Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly Thr 435 440 445Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn Ser 450 455 460Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn Val Val465 470 475 480Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly 485 490 495His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val Gly 500 505 510Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln Arg 515 520 525Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu Glu 530 535 540Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe Gln Ser545 550 555 560Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser 565 570 575Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val Glu 580 585 590Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala Ala 595 600 605Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys 610 615 620Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Ser Arg Leu Asp Lys625 630 635 640Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val Gly Ile 645 650 655Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val Glu Gln 660 665 670Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu Asp Ala 675 680 685Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys Pro Leu 690 695 700Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys Ser Phe705 710 715 720Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His Leu Gly 725 730 735Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln Leu Ala 740 745 750Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr Ala Leu 755 760 765Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp Gln Glu 770 775 780His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp Ser Met785 790 795 800Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln Gly Ala 805 810 815Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly Leu Glu 820 825 830Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu Asp Asp 835 840 845Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu 850 855 860Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu865 870 875 880Pro Gly109817PRTArtificial sequencesynthetic sequence 109Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr65 70 75 80His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser145 150 155 160Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180 185 190Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr225 230 235 240Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met Lys 595 600 605Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys 610 615 620Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu Lys Asn625 630 635 640Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro Leu Thr 645 650 655Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu Glu Gln 660 665 670Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile Leu Lys 675 680 685Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu Phe Val 690 695 700Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala Ser Val705 710 715 720Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser Ala Thr 725 730 735Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu Thr Val 740 745 750Ser Ala Ala Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ala Leu 755 760 765Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 770 775 780Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp785 790 795 800Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly 805 810 815Ser110809PRTArtificial sequencesynthetic sequence 110Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr65 70 75 80His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser145 150 155 160Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180 185

190Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr225 230 235 240Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Leu Leu Glu Ile Arg 595 600 605Ala Ala Phe Leu Arg Gln Arg Asn Thr Ala Leu Arg Thr Glu Val Ala 610 615 620Glu Leu Glu Gln Glu Val Gln Arg Leu Glu Asn Glu Val Ser Gln Tyr625 630 635 640Glu Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys Gly Gly Ser Gly Gly 645 650 655Ser Gly Gly Ser Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp 660 665 670Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys 675 680 685Ala Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr 690 695 700Lys Arg Ser Pro Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg705 710 715 720Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp 725 730 735Leu Asp Met Ile Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu 740 745 750Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr 755 760 765Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln 770 775 780His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly785 790 795 800Gln Arg Gln Leu Thr Val Ser Ala Ala 805111719PRTArtificial sequencesynthetic sequence 111Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gln 20 25 30Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser 35 40 45Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr 50 55 60Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly65 70 75 80Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg 85 90 95Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 100 105 110Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 115 120 125Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr 130 135 140Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met 165 170 175Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser 180 185 190Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro 195 200 205Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly 210 215 220Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser225 230 235 240Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 245 250 255Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys Ala 260 265 270Ser Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro 275 280 285Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp 290 295 300Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly305 310 315 320Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn 325 330 335Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys 340 345 350Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys 355 360 365Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys 370 375 380Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu385 390 395 400Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu 405 410 415Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu 420 425 430Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His 435 440 445Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe 450 455 460Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg465 470 475 480Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly 485 490 495Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile 500 505 510Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln 515 520 525Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala 530 535 540Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser545 550 555 560Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val 565 570 575Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu 580 585 590Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Leu Pro Lys Lys Lys 595 600 605Arg Lys Val Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser 610 615 620Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu625 630 635 640Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 645 650 655Asp Leu Asp Met Leu Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 660 665 670Leu Glu Ile Glu Ala Ala Phe Leu Glu Arg Glu Asn Thr Ala Leu Glu 675 680 685Thr Arg Val Ala Glu Leu Arg Gln Arg Val Gln Arg Leu Arg Asn Arg 690 695 700Val Ser Gln Tyr Arg Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys705 710 715112272PRTHomo sapiens 112Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val1 5 10 15Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys65 70 75 80Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His145 150 155 160Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln225 230 235 240Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 270113551PRTHomo sapiens 113Met Ala Ala Pro Ala Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu1 5 10 15Leu Pro Leu Ala Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser 20 25 30Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 40 45Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 55 60Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser65 70 75 80Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln 85 90 95Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu 100 105 110Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu 115 120 125Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu 130 135 140Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr145 150 155 160Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His 165 170 175Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp 180 185 190Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val 195 200 205Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser 210 215 220Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr225 230 235 240Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 245 250 255Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro 260 265 270Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe 275 280 285Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu 290 295 300Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro305 310 315 320Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu 325 330 335Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn 340 345 350His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His 355 360 365Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr 370 375 380Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro385 390 395 400Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp 405 410 415Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro 420 425 430Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser 435 440 445Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro 450 455 460Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro465 470 475 480Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala 485 490 495Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro 500 505 510Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540Gln Asp Pro Thr His Leu Val545 550114369PRTHomo sapiens 114Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu1 5 10 15Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40 45Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50 55 60Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro65 70 75 80Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn 85 90 95Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr 100 105 110Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe 115 120 125Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln 130 135 140Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu145 150 155 160Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170 175Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp 180 185 190Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe 195 200 205Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg 210 215 220Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp225 230 235 240Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe 245 250 255Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu 260 265 270Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro 275 280 285Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His 290 295

300Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser305 310 315 320Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro 325 330 335Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn 340 345 350Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu 355 360 365Thr

Claims

1. A genetically modified, in vitro immune cell, wherein the immune cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding: a) a chimeric polypeptide comprising: i) an antibody specific for a target antigen; and ii) a binding triggered transcriptional activator; and b) a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell, wherein the nucleotide sequence encoding the cytokine or proliferation-inducing polypeptide is operably linked to a transcriptional control element responsive to the transcriptional activator.

2. The genetically modified immune cell of claim 1, wherein the immune cell is a T cell or a macrophage.

3. The genetically modified immune cell of claim 1, wherein the binding triggered transcriptional activator comprises, from N-terminal to C-terminal and in covalent linkage: i) an extracellular domain comprising an antibody specific for a target antigen; ii) a Notch regulatory polypeptide that comprises one or more proteolytic cleavage sites; and iii) an intracellular domain comprising a transcriptional activator, wherein binding of the antibody to the target antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain

4. The genetically modified immune cell of claim 3, wherein the Notch regulatory region comprises a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site.

5. The genetically modified immune cell of claim 3 or claim 4, wherein the Notch regulatory region further comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats.

6. The genetically modified immune cell of claim 1, wherein the binding triggered transcriptional activator comprises, from N-terminal to C-terminal and in covalent linkage: a) an extracellular domain comprising an antibody specific for a target antigen; b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site; c) a cleavable transmembrane domain; and d) an intracellular domain comprising a Notch intracellular signaling domain comprising a transcriptional activator, wherein binding of the antibody to the target antigen induces cleavage of the non-Notch force sensor cleavage domain at the proteolytic cleavage site, thereby releasing the intracellular domain, and wherein the non-Notch force sensor cleavage domain is selected from the group consisting of: a von Willebrand Factor (vWF) cleavage domain, an amyloid-beta cleavage domain, a CD16 cleavage domain, a CD44 cleavage domain, a Delta cleavage domain, a cadherin cleavage domain, an ephrin-type receptor or ephrin ligand cleavage domain, a protocadherin cleavage domain, a filamin cleavage domain, a synthetic E cadherin cleavage domain, an interleukin-1 receptor type 2 (IL1R2) cleavage domain, a major prion protein (PrP) cleavage domain, a neuregulin cleavage domain and an adhesion-GPCR cleavage domain,

7. The genetically modified immune cell of claim 6, wherein the non-Notch force sensor cleavage domain is a vWF cleavage domain

8. The genetically modified immune cell of claim 7, wherein the vWF cleavage domain comprises a vWF A2 domain or a variant thereof.

9. The genetically modified immune cell of any one of claims 1-8, wherein the antibody is a nanobody, a diabody, a triabody, or a minibody, a F(ab').sub.2 fragment, a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody (sdAb).

10. The genetically modified immune cell of any one of claims 1-9, wherein the cytokine is IL-2.

11. The genetically modified immune cell of claim 10, wherein the IL-2 is an IL-2 variant that exhibits increased binding affinity for IL-2R.beta. compared to wild-type IL-2.

12. The genetically modified T cell of claim 11, wherein the IL-2 variant comprises amino acid substitutions L80F, R81D, L85V, I86V, and I92F, compared to wild-type human IL-2.

13. The genetically modified immune cell of claim 10, wherein the IL-2 is a variant that preferentially activates regulatory T cells (T regs).

14. The genetically modified immune cell of claim 10, wherein the IL-2 is a variant that preferentially activates natural killer (NK) cells.

15. The genetically modified immune cell of claim 10, wherein the IL-2 is: i) a variant IL-2 that binds to a variant IL-2R.beta. comprising one or more amino acid substitutions selected from Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R; and ii) exhibits reduced binding to wild-type IL-2R.beta..

16. The genetically modified immune cell of claim 15, wherein the variant IL-2 comprises one or more amino acid substitutions selected from: i) H16N, L19V, D2ON, Q22T, M23H, and G27K; ii) E15D, H16N, L19V, D2OL, Q22T, and M23H; iii) E15D, H16N, L19V, D2OL, Q22T, and M23A; or iv) E15D, H16N, L19V, D2OL, Q22K, M23A.

17. The genetically modified immune cell of any one of claims 1-9, wherein the cytokine is IL-15 or IL-7.

18. The genetically modified immune cell of any one of claims 1-9, wherein the proliferation-inducing polypeptide binds IL-2R.beta..gamma..sub.c heterodimer, but does not bind IL-2R.alpha. or IL-2R.beta..

19. The genetically modified immune cell of any one of claims 1-18, wherein the target antigen is a cancer-associated antigen.

20. The genetically modified immune cell of claim 19, wherein the cancer-associated antigen is selected from CD19, CD20, CD38, CD30, Her2/neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, and GD2.

21. The genetically modified immune cell of any one of claims 1-18, wherein the target antigen is tissue-specific antigen or an organ-specific antigen or a cell type-specific antigen.

22. The genetically modified immune cell of any one of claims 1-18, wherein the target antigen is a stromal cell antigen.

23. The genetically modified immune cell of any one of claims 1-22, wherein the genetically modified immune cell is a T cell that does not express an endogenous major histocompatibility complex (MHC) class I polypeptide on its surface.

24. The genetically modified immune cell of claim 23, wherein the genetically modified immune cell comprises a deletion of all or a portion of at least one MHC class I coding region.

25. The genetically modified immune cell of claim 24, wherein the at least one MHC class I coding region is a .beta.2-microglobulin coding region.

26. The genetically modified immune cell of any one of claims 1-25, wherein the genetically modified immune cell is a T cell that does not express an endogenous T-cell receptor (TCR).

27. A composition comprising: a) the genetically modified immune cell of any one of claims 1-26; and b) a cytotoxic T cell (CTL).

28. The composition of claim 27, wherein the CTL is genetically modified to express: a) an exogenous T-cell receptor (TCR), wherein the exogenous TCR is specific for a target antigen; or b) a chimeric antigen receptor (CAR), wherein the CAR is specific for a target antigen; or c) a bispecific T-cell engager (BiTE), wherein the BiTE comprises: i) a first antigen-binding region specific for CD3; and ii) a second antigen-binding region specific for a target antigen other than CD3.

29. The composition of claim 27 or claim 28, wherein the TCR, the CAR, or the BiTE is specific for a cancer-associated antigen, and wherein the antibody present in the chimeric polypeptide is specific for a cancer-associated antigen.

30. The composition of claim 29, wherein the TCR, the CAR, or the BiTE is specific for the same cancer-associated antigen as the cancer-associate antigen to which the antibody present in the chimeric polypeptide binds.

31. The composition of claim 29, wherein the TCR, the CAR, or the BiTE is specific for a cancer-associated antigen that is different from the cancer-associate antigen to which the antibody present in the chimeric polypeptide binds.

32. The composition of any one of claims 28-31, wherein the CTL is genetically modified to express a CAR, and wherein the CAR comprises: a) an extracellular domain comprising the antigen-binding domain; b) a transmembrane region; and c) an intracellular signaling domain

33. The composition of claim 32, wherein the intracellular signaling domain comprises: i) a signaling domain from the zeta chain of human CD3; and ii) one or more costimulatory polypeptides.

34. The composition of claim 33, wherein the one or more costimulatory polypeptides is selected from CD28, 4-1BB, and OX-40.

35. The composition of any one of claims 32-34, wherein the CAR is a single polypeptide chain.

36. The composition of any one of claims 32-34, wherein the CAR comprises 2 polypeptide chains.

37. The composition of claim 36, wherein the 2 polypeptide chains dimerize in the presence of a small molecule dimerizer.

38. A method of increasing proliferation and/or activity of a target immune cell in an individual, the method comprising administering to the individual a genetically modified immune cell according to any one of claims 1-26 or a composition according to any one of claims 27-37.

39. The method of claim 38, wherein the target immune cell is a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a natural killer (NK) cell, or a regulatory T cell (Treg).

40. The method of claim 38 or claim 39, wherein the target immune cell is an endogenous immune cell.

41. The method of claim 38 or claim 39, wherein the target immune cell is an exogenous immune cell that has been genetically modified and introduced into the individual.

42. The method of claim 41, wherein the target immune cell is T cell that has been genetically modified to express an exogenous T-cell receptor (TCR) or an exogenous chimeric antigen receptor (CAR).

43. The method of claim 41 or claim 42, wherein the target immune cell is genetically modified to express a variant IL2 receptor on its surface.

44. The method of any one of claims 38-43, wherein the target immune cell is specific for a cancer-associated antigen.

45. The method of claim 44, wherein the target immune cell is specific for the same cancer-associated antigen to which the antibody present in the chimeric polypeptide binds.

46. The method of any one of claims 38-45, comprising administering to the individual an effective amount of a cytokine or proliferation-inducing polypeptide that increases proliferation and/or activity of an effector immune cell.

47. The method of claim 46, wherein the cytokine is IL-2.

48. The method of claim 47, wherein the IL-2 is an IL-2 variant that exhibits increased binding affinity for IL-2R13 compared to wild-type IL-2.

49. The method of claim 48, wherein the IL-2 variant comprises amino acid substitutions L80F, R81D, L85V, I86V, and I92F, compared to wild-type human IL-2.

50. The method of claim 47, wherein the IL-2 is a variant that preferentially activates regulatory T cells (T regs).

51. The method of claim 47, wherein the IL-2 is a variant that preferentially activates natural killer (NK) cells.

52. The method of claim 47, wherein the IL-2 is: i) a variant IL-2 that binds to a variant IL-2R.beta. comprising one or more amino acid substitutions selected from Q70Y, T73D, T73Y, H133D, H133E, H133K, Y134F, Y134E, and Y134R; and ii) exhibits reduced binding to wild-type IL-2R.beta..

53. The method of claim 52, wherein the variant IL-2 comprises one or more amino acid substitutions selected from: i) H16N, L19V, D2ON, Q22T, M23H, and G27K; ii) E15D, H16N, L19V, D20L, Q22T, and M23H; iii) E15D, H16N, L19V, D20L, Q22T, and M23A; or iv) E15D, H16N, L19V, D20L, Q22K, M23A.

54. The method of claim 46, wherein the cytokine is IL-15 or IL-7.

55. The method of claim 46, wherein the proliferation-inducing polypeptide binds IL-2R.beta..gamma..sub.c heterodimer, but does not bind IL-2R.alpha. or IL-2R.beta..